Hiv pre-immunization and immunotherapy

ABSTRACT

The present invention relates generally to immunization and immunotherapy for the treatment or prevention of HIV. In particular, the methods include in vivo and/or ex vivo enrichment of HIV-specific CD4+ T cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to: U.S. Provisional Patent ApplicationNo. 62/360,185 filed on Jul. 8, 2016 entitled “HIV PRE-IMMUNIZATION ANDIMMUNOTHERAPY”, U.S. Provisional Patent Application No. 62/385,864 filedon Sep. 9, 2016 entitled “HIV PRE-IMMUNIZATION AND IMMUNOTHERAPY”, U.S.Provisional Patent Application No. 62/409,270 filed on Oct. 17, 2016entitled “HIV PRE-IMMUNIZATION AND IMMUNOTHERAPY,” and PCT/US17/13019filed on Jan. 11, 2017 entitled “HIV PRE-IMMUNIZATION ANDIMMUNOTHERAPY”, the disclosures of which are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates generally to the field of immunization andimmunotherapy for the treatment and prevention of HIV. In particular,the disclosed methods relate to obtaining and processing leukocytes fromHIV+ individuals seeking a functional cure to prepare a cell productsuitable for infusion to such HIV+ individuals.

BACKGROUND OF THE INVENTION

Combination antiretroviral therapy (cART) (also known as Highly ActiveAntiretroviral Therapy or HAART) limits HIV-1 replication and slowsdisease progression, but drug toxicities and the emergence ofdrug-resistant viruses are challenges for long-term control inHIV-infected persons. Additionally, traditional antiretroviral therapy,while successful at delaying the onset of AIDS or death, has yet toprovide a functional cure. Alternative treatment strategies are needed.

Intense interest in immunotherapy for HIV infection has beenprecipitated by emerging data indicating that the immune system has amajor, albeit usually insufficient, role in limiting HIV replication.Virus-specific T-helper cells, which are critical to maintenance ofcytolytic T cell (CTL) function, likely play a role. Viremia is alsoinfluenced by neutralizing antibodies, but they are generally low inmagnitude in HIV infection and do not keep up with evolving viralvariants in vivo.

Together these data indicate that increasing the strength and breadth ofHIV-specific cellular immune responses may have a clinical benefitthrough so-called HIV immunotherapy. Some studies have tested vaccinesagainst HIV, but success has been limited to date. Additionally, therehas been interest in augmenting HIV immunotherapy by utilizing genetherapy techniques, but as with other immunotherapy approaches, successhas been limited.

Viral vectors can be used to transduce genes into target cells owing tospecific virus-host cell interactions and mechanisms for expressingtherapeutic gene constructs. As a result, viral vectors have been usedas vehicles for the transfer of genes into many different cell typesincluding whole T cells or other immune cells as well as embryos,fertilized eggs, isolated tissue samples, tissue targets in situ andcultured cells. The ability to introduce and express foreign or alteredgenes in a cell is useful for therapeutic interventions such as genetherapy, somatic cell reprogramming of induced pluripotent stem cells,and various types of immunotherapy.

Gene therapy is one of the ripest areas of biomedical research with thepotential to create new therapeutics that may involve the use of viralvectors. In view of the wide variety of potential genes available fortherapy, an efficient means of delivering these genes is needed tofulfill the promise of gene therapy as a means of treating infectiousand non-infectious diseases. Several viral systems including murineretrovirus, adenovirus, parvovirus (adeno-associated virus), coxsackievirus, vaccinia virus, and herpes virus have been proposed astherapeutic gene transfer vectors.

There are many factors that must be considered when developing viralvectors including tissue tropism, stability of virus preparations,stability and control of expression, genome packaging capacity,construct-dependent vector stability, and whether or not the desiredoutcome is to have stable gene integration into the host genome. Inaddition, in vivo application of viral vectors is often limited by hostimmune responses against viral structural proteins and/or transducedgene products.

Thus, toxicity and safety are key hurdles that must be overcome forviral vectors to be used in vivo for the treatment of subjects. Thereare numerous historical examples of gene therapy applications in humansthat have met with problems associated with the host immune responsesagainst the gene delivery vehicles or the therapeutic gene products.Viral vectors (e.g., adenovirus) which co-transduce several viral genestogether with one or more therapeutic gene(s) are particularlyproblematic.

Although lentiviral vectors do not generally induce cytotoxicity and donot elicit strong host immune responses, some lentiviral vectors such asHIV-1, which encode several immunostimulatory gene products, have thepotential to cause cytotoxicity and induce strong immune responses invivo. However, this may not be a concern for lentivirus-derivedtransducing vectors that do not encode viral genes after transduction.Of course, this may not always be the case, as sometimes the purpose ofthe vector is to encode a protein that will provoke a clinically usefulimmune response.

Another important issue related to the use of lentiviral vectors is thatof possible cytopathogenicity upon exposure to some cytotoxic viralproteins. Exposure to certain HIV-1 proteins may induce cell death orfunctional unresponsiveness in T cells. Likewise, the possibility ofgenerating replication-competent, virulent virus by recombination isoften a concern. Accordingly, there remains a need for improvedtreatments of HIV.

SUMMARY OF THE INVENTION

In one aspect, a method of treating cells infected with HIV is provided.The method variously includes contacting peripheral blood mononuclearcells (PBMC) isolated from a subject infected with HIV with atherapeutically effective amount of a stimulatory agent, wherein thecontacting is carried out ex vivo; transducing the PBMC ex vivo with aviral delivery system encoding at least one genetic element; andculturing the transduced PBMC for a sufficient period of time to ensureadequate transduction. In embodiments, the transduced PBMC are culturedfrom about 1 to about 35 days. In embodiments, the method furtherincludes infusing the transduced PBMC into a subject. In embodiments,the method further includes positively selecting HIV-specific CD4+ Tcells from the PBMC. In further embodiments, the HIV-specific CD4+ Tcells are positively selected using at least one physical method ofselection. In embodiments, the subject is a human. In embodiments, thestimulatory agent includes any agent suitable for stimulating a T cellresponse in a subject. In embodiments, the stimulatory agent is apeptide or mixture of peptides, and in embodiments includes a gagpeptide. In further embodiments, the stimulatory agent is a vaccine. Inembodiments, the vaccine is a HIV vaccine, and in embodiments, the HIVvaccine is a MVA/HIV62B vaccine or a variant thereof. In embodiments,the viral delivery system includes a lentiviral particle. Inembodiments, the at least one genetic element includes a small RNAcapable of inhibiting production of chemokine receptor CCR5. In furtherembodiments, the at least one genetic element includes at least onesmall RNA capable of targeting a HIV RNA sequence. In furtherembodiments, the at least one genetic element includes a small RNAcapable of inhibiting production of chemokine receptor CCR5 and at leastone small RNA capable of targeting an HIV RNA sequence. The HIV RNAsequence includes any HIV sequence suitable for targeting by a viraldelivery system. In embodiments, the HIV RNA sequence includes one ormore of a HIV Vif sequence, a HIV Tat sequence, or a variant thereof.The at least one genetic element includes any genetic element capable ofbeing expressed by a viral delivery system. In embodiments, the at leastone genetic element includes a microRNA or a shRNA. In furtherembodiments, the at least one genetic element comprises a microRNAcluster.

In another aspect, a method is disclosed that includes obtainingperipheral blood from HIV+ individuals; fractionating the blood toobtain a PBMC population; contacting the PBMC population with purifiedantigen-presenting cells or peptides or proteins representing componentsof HIV; culturing the contacted PBMC population for about 1 to about 35days to increase the number of antigen-specific T cells; positivelyselecting cells that respond to peptide stimulation to produce anenriched cell fraction; transducing the enriched cell fraction ex vivowith a viral delivery system as detailed herein, and culturing thetransduced cell fraction for a period of time sufficient to ensureadequate transduction. The order of the method steps disclosed hereincan be changed. As a non-limiting example, the steps of positivelyselecting cells and transducing cells may be reversed and polyclonalstimulation may also be added to improve transduction efficiency.

In embodiments, the PBMC population is further purified to produce apurified fraction of PBMC. In embodiments, further purified fractions ofPBMC are contacted with peptides or proteins representing components ofHIV.

In another aspect, the at least one genetic element includes a microRNAhaving at least 80%, or at least 85%, or at least 90%, or at least 95%percent identity withAGGTATATTGCTGTTGACAGTGAGCGACTGTAAACTGAGCTTGCTCTACTGTGAAGCCACAGATGGGTAGAGCAAGCACAGTTTACCGCTGCCTACTGCCTCGGACTTCAA GGGGCTT (SEQ IDNO: 1). In embodiments, the at least one genetic element comprises SEQID NO: 1.

In another aspect, the at least one genetic element includes a microRNAhaving at least 80%, or at least 85%, or at least 90%, or at least 95%percent identity withCATCTCCATGGCTGTACCACCTTGTCGGGGGATGTGTACTTCTGAACTTGTGTTGAATCTCATGGAGTTCAGAAGAACACATCCGCACTGACATTTTGGTATCTTTCATCTG ACCA (SEQ IDNO: 2); or at least 80%, or at least 85%, or at least 90%, or at least95% percent identity withGGGCCTGGCTCGAGCAGGGGGCGAGGGATTCCGCTTCTTCCTGCCATAGCGTGGTCCCCTCCCCTATGGCAGGCAGAAGCGGCACCTTCCCTCCCAATGACCGCGTCTTC GTCG (SEQ IDNO: 3). In embodiments, the at least one genetic element includes SEQ IDNO: 2; or SEQ ID NO: 3.

In another aspect, the microRNA cluster includes a sequence having atleast 80%, or at least 85%, or at least 90%, or at least 95% percentidentity with AGGTATATTGCTGTTGACAGTGAGCGACTGTAAACTGAGCTTGCTCTACTGTGAAGCCACAGATGGGTAGAGCAAGCACAGTTTACCGCTGCCTACTGCCTCGGACTTCAAGGGGCTTCCCGGGCATCTCCATGGCTGTACCACCTTGTCGGGGGATGTGTACTTCTGAACTTGTGTTGAATCTCATGGAGTTCAGAAGAACACATCCGCACTGACATTTTGGTATCTTTCATCTGACCAGCTAGCGGGCCTGGCTCGAGCAGGGGGCGAGGGATTCCGCTTCTTCCTGCCATAGCGTGGTCCCCTCCCCTATGGCAGGCAGAAGCGGCACCTTCCCTCCCAATGACCGCGTCTTCGTC (SEQ ID NO: 31). In embodiments, the microRNAcluster includes SEQ ID NO: 31.

In another aspect, a method of treating HIV infection in a subject isdisclosed. The method variously includes immunizing the subject with aneffective amount of a first stimulatory agent; removing leukocytes fromthe subject and obtaining peripheral blood mononuclear cells (PBMC). Themethod further includes contacting the PBMC ex vivo with atherapeutically effective amount of a second stimulatory agent;transducing the PBMC ex vivo with a viral delivery system encoding atleast one genetic element; and culturing the transduced PBMC for asufficient period of time to ensure adequate transduction. Inembodiments, the transduced PBMC may be cultured from about 1 to about35 days. In embodiments, the method further includes positivelyselecting HIV-specific CD4+ T cells from the PBMC. In furtherembodiments, the HIV-specific CD4+ T cells are positively selected usingat least one physical method of selection. In embodiments, the methodfurther involves infusing the transduced PBMC into a subject. Thesubject may be a human. The first and second stimulatory agents may bethe same or different. The first and second stimulatory agents mayinclude one or more of a peptide or mixture of peptides. In embodiments,at least one of the first and second stimulatory agents includes a gagpeptide. In embodiments, at least one of the first and secondstimulatory agents includes a mixture of gag peptides that arerecognized by immune cells resident in the PBMC or in the purifiedfractions of PBMC. The at least one of the first and second stimulatoryagents may include a vaccine. In embodiments, the vaccine is a HIVvaccine; in further embodiments, the HIV vaccine is a MVA/HIV62B vaccineor a variant thereof. In embodiments, the viral delivery system includesa lentiviral particle. In embodiments, the at least one genetic elementincludes a small RNA capable of inhibiting production of chemokinereceptor CCR5. In embodiments, the at least one genetic element includesat least one small RNA capable of targeting an HIV RNA sequence. Inembodiments, the at least one genetic element includes a small RNAcapable of inhibiting production of chemokine receptor CCR5 and at leastone small RNA capable of targeting an HIV RNA sequence. The HIV RNAsequence may include a HIV Vif sequence, a HIV Tat sequence, or avariant thereof. The at least one genetic element may include a microRNAor a shRNA. In embodiments, the at least one genetic element comprises asynthetic microRNA cluster designed for simultaneous expression of allelements in the cluster under control of a single transcriptionalpromoter. In embodiments, the PBMC population is further purified toproduce a purified fraction of PBMC.

In another aspect, the at least one genetic element includes a microRNAhaving at least 80%, or at least 85%, or at least 90%, or at least 95%percent identity withAGGTATATTGCTGTTGACAGTGAGCGACTGTAAACTGAGCTTGCTCTACTGTGAAGCCACAGATGGGTAGAGCAAGCACAGTTTACCGCTGCCTACTGCCTCGGACTTCAA GGGGCTT (SEQ IDNO: 1). In embodiments, the at least one genetic element comprises SEQID NO: 1.

In another aspect, the at least one genetic element includes a microRNAhaving at least 80%, or at least 85%, or at least 90%, or at least 95%percent identity withCATCTCCATGGCTGTACCACCTTGTCGGGGGATGTGTACTTCTGAACTTGTGTTGAATCTCATGGAGTTCAGAAGAACACATCCGCACTGACATTTTGGTATCTTTCATCTG ACCA (SEQ IDNO: 2); or at least 80%, or at least 85%, or at least 90%, or at least95% percent identity withGGGCCTGGCTCGAGCAGGGGGCGAGGGATTCCGCTTCTTCCTGCCATAGCGTGGTCCCCTCCCCTATGGCAGGCAGAAGCGGCACCTTCCCTCCCAATGACCGCGTCTTC GTCG (SEQ IDNO: 3). In embodiments, the at least one genetic element includes SEQ IDNO: 2 or SEQ ID NO: 3.

In another aspect, the microRNA cluster includes a sequence having atleast 80%, or at least 85%, or at least 90%, or at least 95% percentidentity with AGGTATATTGCTGTTGACAGTGAGCGACTGTAAACTGAGCTTGCTCTACTGTGAAGCCACAGATGGGTAGAGCAAGCACAGTTTACCGCTGCCTACTGCCTCGGACTTCAAGGGGCTTCCCGGGCATCTCCATGGCTGTACCACCTTGTCGGGGGATGTGTACTTCTGAACTTGTGTTGAATCTCATGGAGTTCAGAAGAACACATCCGCACTGACATTTTGGTATCTTTCATCTGACCAGCTAGCGGGCCTGGCTCGAGCAGGGGGCGAGGGATTCCGCTTCTTCCTGCCATAGCGTGGTCCCCTCCCCTATGGCAGGCAGAAGCGGCACCTTCCCTCCCAATGACCGCGTCTTCGTC (SEQ ID NO: 31). In embodiments, the microRNAcluster includes SEQ ID NO: 31.

In another aspect, a lentiviral vector is disclosed. The lentiviralvector includes at least one encoded genetic element, wherein the atleast one encoded genetic element comprises a small RNA capable ofinhibiting production of chemokine receptor CCR5. The at least oneencoded genetic element may also comprise at least one small RNA capableof targeting an HIV RNA sequence. In another aspect, the at least oneencoded genetic element comprises a small RNA capable of inhibitingproduction of chemokine receptor CCR5 and at least one small RNA capableof targeting an HIV RNA sequence. The HIV RNA sequence may include a HIVVif sequence, a HIV Tat sequence, or a variant thereof. The at least oneencoded genetic element may include a microRNA or a shRNA. The at leastone encoded genetic element may include a synthetic microRNA clusterdesigned for simultaneous expression of all elements in the clusterunder control of a single transcriptional promoter.

In another aspect, the at least one genetic element includes a microRNAhaving at least 80%, or at least 85%, or at least 90%, or at least 95%percent identity withAGGTATATTGCTGTTGACAGTGAGCGACTGTAAACTGAGCTTGCTCTACTGTGAAGCCACAGATGGGTAGAGCAAGCACAGTTTACCGCTGCCTACTGCCTCGGACTTCAA GGGGCTT (SEQ IDNO: 1). In embodiments, the at least one genetic element comprises SEQID NO: 1.

In another aspect, the at least one genetic element includes a microRNAhaving at least 80%, or at least 85%, or at least 90%, or at least 95%percent identity withCATCTCCATGGCTGTACCACCTTGTCGGGGGATGTGTACTTCTGAACTTGTGTTGAATCTCATGGAGTTCAGAAGAACACATCCGCACTGACATTTTGGTATCTTTCATCTG ACCA (SEQ IDNO: 2); or at least 80%, or at least 85%, or at least 90%, or at least95% percent identity withGGGCCTGGCTCGAGCAGGGGGCGAGGGATTCCGCTTCTTCCTGCCATAGCGTGGTCCCCTCCCCTATGGCAGGCAGAAGCGGCACCTTCCCTCCCAATGACCGCGTCTTC GTCG (SEQ IDNO: 3). In embodiments, the at least one genetic element includes SEQ IDNO: 2; or SEQ ID NO: 3.

In another aspect, the microRNA cluster includes a sequence having atleast 80%, or at least 85%, or at least 90%, or at least 95% percentidentity with AGGTATATTGCTGTTGACAGTGAGCGACTGTAAACTGAGCTTGCTCTACTGTGAAGCCACAGATGGGTAGAGCAAGCACAGTTTACCGCTGCCTACTGCCTCGGACTTCAAGGGGCTTCCCGGGCATCTCCATGGCTGTACCACCTTGTCGGGGGATGTGTACTTCTGAACTTGTGTTGAATCTCATGGAGTTCAGAAGAACACATCCGCACTGACATTTTGGTATCTTTCATCTGACCAGCTAGCGGGCCTGGCTCGAGCAGGGGGCGAGGGATTCCGCTTCTTCCTGCCATAGCGTGGTCCCCTCCCCTATGGCAGGCAGAAGCGGCACCTTCCCTCCCAATGACCGCGTCTTCGTC (SEQ ID NO: 31). In embodiments, the microRNAcluster includes SEQ ID NO: 31.

In another aspect, a lentiviral vector system for expressing alentiviral particle is disclosed. The system includes a lentiviralvector as described herein; an envelope plasmid for expressing anenvelope protein preferably optimized for infecting a cell; and at leastone helper plasmid for expressing genes of interest. In embodiments, thegenes of interest include one or more of gag, pol, and rev genes. Inembodiments, the lentiviral vector, the envelope plasmid, and the atleast one helper plasmid are transfected into a packaging cell line. Infurther embodiments, a lentiviral particle is produced by the packagingcell line. In embodiments, the lentiviral particle is capable ofmodulating production of a target of interest. In embodiments, thetarget of interest is any of chemokine receptor CCR5 or an HIV RNAsequence. The system may further include a first helper plasmid and asecond helper plasmid. In embodiments, a first helper plasmid expressesthe gag and pol genes, and a second helper plasmid expresses the revgene.

In another aspect, a lentiviral particle capable of infecting a cell isprovided. The lentiviral particle includes an envelope proteinpreferably optimized for infecting a cell, and a lentiviral vector asdescribed herein. In embodiments, the envelope protein may be optimizedfor infecting a T cell. In embodiments, the envelope protein isoptimized for infecting a CD4+ T cell.

In another aspect, a modified cell is provided. The modified cellincludes any cell capable of being infected with a lentiviral vectorsystem for use in accordance with present aspects and embodiments. Inembodiments, the cell is a CD4+ T cell that is infected with alentiviral particle. In embodiments, the CD4+ T cell also has beenselected to recognize an HIV antigen. In embodiments, the HIV antigenincludes a gag antigen. In embodiments, the CD4+ T cell expresses adecreased level of CCR5 following infection with the lentiviralparticle.

In another aspect, a method of selecting a subject for a therapeutictreatment regimen is provided. The method variously includes immunizingthe subject with an effective amount of a first stimulatory agent;removing leukocytes from the subject and purifying peripheral bloodmononuclear cells (PBMC) and determining a first quantifiablemeasurement associated with at least one factor associated with thePBMC; contacting the PBMC ex vivo with a therapeutically effectiveamount of a second stimulatory agent, and determining a secondmeasurement associated with the at least one factor associated with thePBMC, whereby when the second quantifiable measurement is higher thanthe first quantifiable measurement, the subject is selected for thetreatment regimen. The at least one factor may include any factorassociated with T cell proliferation or IFN gamma production.

The foregoing general description and following brief description of thedrawings and detailed description are exemplary and explanatory and areintended to provide further explanation of the invention as claimed.Other objects, advantages, and novel features will be readily apparentto those skilled in the art from the following brief description of thedrawings and detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a flow diagram of an ex vivo treatment method of thepresent disclosure.

FIG. 2 depicts CD4+ T cell alteration and prevention of new infection inaccordance with the present disclosure.

FIG. 3 depicts an exemplary lentiviral vector system comprised of atherapeutic vector, a helper plasmid, and an envelope plasmid. Thetherapeutic vector shown here is a preferred therapeutic vector, whichis also referred to herein as AGT103, and containsmiR30CCR5-miR21Vif-miR185-Tat.

FIG. 4 depicts an exemplary 3-vector lentiviral vector system in acircularized form.

FIG. 5 depicts an exemplary 4-vector lentiviral vector system in acircularized form.

FIG. 6 depicts a further exemplary 3-vector lentiviral vector system ina circularized form.

FIG. 7 depicts exemplary vector sequences. Positive (i.e., genomic)strand sequences of the promoter and miR cluster were developed forinhibiting the spread of CCR5-tropic HIV strains. Sequences that are notunderlined comprise the EF-1alpha promoter of transcription that wasselected as being a preferable promoter for this miR cluster. Sequencesthat are underlined show the miR cluster consisting of miR30 CCR5, miR21Vif, and miR185 Tat (as shown collectively in SEQ ID NO: 33).

FIG. 8 depicts exemplary lentiviral vector constructs according tovarious aspects of this disclosure.

FIG. 9 shows knockdown of CCR5 by an experimental vector andcorresponding prevention of R5-tropic HIV infection in AGTc120 cells.(A) shows CCR5 expression in AGTc120 cells with or without AGT103lentivirus vector. (B) shows the sensitivity of transduced AGTc120 cellsto infection with a HIV BaL virus stock that was expressing greenfluorescent protein (GFP) fused to the Nef gene of HIV.

FIG. 10 depicts data demonstrating regulation of CCR5 expression byshRNA inhibitor sequences in a lentiviral vector of the presentdisclosure. (A) Screening data for potential candidates is shown. (B)CCR5 knock-down data following transduction with CCR5 shRNA-1 (SEQ IDNO: 16) is shown.

FIG. 11 depicts data demonstrating regulation of HIV components by shRNAinhibitor sequences in a lentiviral vector of the present disclosure.(A) Knock-down data for the rev/tat target gene is shown. (B) Knock-downdata for the gag target gene is shown.

FIG. 12 depicts data demonstrating that AGT103 reduces expression of Tatprotein expression in cells transfected with an HIV expression plasmid,as described herein.

FIG. 13 depicts data demonstrating regulation of HIV components bysynthetic microRNA sequences in a lentiviral vector of the presentdisclosure. (A) Tat knock-down data is shown. (B) Vif knock-down data isshown.

FIG. 14 depicts data demonstrating regulation of CCR5 expression bysynthetic microRNA sequences in a lentiviral vector of the presentdisclosure.

FIG. 15 depicts data demonstrating regulation of CCR5 expression bysynthetic microRNA sequences in a lentiviral vector of the presentdisclosure containing either a long or short WPRE sequence.

FIG. 16 depicts data demonstrating regulation of CCR5 expression bysynthetic microRNA sequences in a lentiviral vector of the presentdisclosure with or without a WPRE sequence.

FIG. 17 depicts data demonstrating regulation of CCR5 expression by aCD4 promoter regulating synthetic microRNA sequences in a lentiviralvector of the present disclosure.

FIG. 18 depicts data demonstrating detection of HIV Gag-specific CD4 Tcells.

FIG. 19 depicts data demonstrating HIV-specific CD4 T cell expansion andlentivirus transduction. (A) An exemplary schedule of treatment isshown. (B) IFN-gamma production in CD4-gated T cells is shown, asdescribed herein. (C) IFN-gamma production and GFP expression inCD4-gated T cells is shown, as described herein. (D) Frequency ofHIV-specific CD4+ T cells is shown, as described herein. (E) IFN-gammaproduction from PBMCs post-vaccination and following ex vivo peptide andexpansion is shown, as described herein.

FIG. 20 depicts data demonstrating a functional assay for a doseresponse of increasing AGT103-GFP and inhibition of CCR5 expression. (A)Dose response data for increasing amounts of AGT103-GFP is shown. (B)Normally distributed populations in terms of CCR5 expression are shownand a left shift of the population average indicates decreasing CCR5expression due to AGT103-GFP transduction. (C) Percentage inhibition ofCCR5 expression with increasing doses of AGT103-GFP is shown.

FIG. 21 depicts data demonstrating AGT103 transduction efficiency forprimary human CD4+ T cells. (A) Frequency of transduced cells(GFP-positive) is shown by FACS, as described herein. (B) Number ofvector copies per cell is shown, as described herein.

FIG. 22 depicts data demonstrating AGT103 inhibition of HIV replicationin primary CD4+ T cells, as described herein.

FIG. 23 depicts data demonstrating AGT103 protection of primary humanCD4⁺ T cells from HIV-induced depletion.

FIG. 24 depicts data demonstrating generation of a CD4+ T cellpopulation that is highly enriched for HIV-specific, AGT103-transducedCD4 T cells. (A) shows CD4 and CD8 expression profiles for cellpopulations, as described herein. (B) shows CD4 and CD8 expressionprofiles for cell populations, as described herein. (C) shows IFN-gammaand CD4 expression profiles for cell populations, as described herein.(D) shows IFN-gamma and GFP expression profiles for cell populations, asdescribed herein.

DETAILED DESCRIPTION Overview

Disclosed herein are methods and compositions for treating and/orpreventing human immunodeficiency virus (HIV) disease to achieve afunctional cure. The methods and compositions include integratinglentivirus, non-integrating lentivirus, and related viral vectortechnology as described below.

Disclosed herein are therapeutic viral vectors (e.g., lentiviralvectors), immunotherapies, and methods for their use for treating HIVinfection. In embodiments, methods and compositions for achieving afunctional cure for HIV infection are provided. As depicted in FIG. 1herein, the various aspects and embodiments include a first stimulationevent, for example a first therapeutic immunization with vaccinesintended to produce strong immune responses against HIV in HIV-infectedpatients, for example with stable suppression of viremia due to dailyadministration of HAART. In embodiments, the first stimulation eventenriches the fraction of HIV-specific CD4 T cells. This is followed by(1) isolating peripheral leukocytes by leukapheresis or purifying PBMCor purifying fractions of PBMC from venous blood, (2) a secondstimulating event, for example stimulating CD4 T cells ex vivo with asuitable stimulatory agent, such as any vaccine or protein, for example,HIV or HIV-related peptides, (3) selecting an enriched cell populationbased on a biological response to peptide stimulation, (4) performingtherapeutic lentivirus transduction, ex vivo T cell culture, and (5)re-infusion back into the original patient.

The various methods and compositions can be used to prevent new cells,such as CD4+ T cells, from becoming infected with HIV. For example asillustrated in FIG. 2, to prevent new cells from becoming infected, CCR5expression can be targeted to prevent virus attachment. Further,destruction of any residual infecting viral RNA can also be targeted. Inrespect of the foregoing, and in reference to FIG. 2 herein,compositions and methods are provided to stop the HIV viral cycle incells that have already become infected with HIV. To stop the HIV viralcycle, viral RNA produced by latently-infected cells, such aslatently-infected CD4+ T cells, is targeted.

Previous efforts to achieve a cure for HIV have fallen short due to,among others, the failure to obtain sufficient numbers of HIV-specificCD4 T cells with protective genetic modifications. When this number isbelow a critical threshold, a functional cure as described herein is notachieved. For example, upon termination of antiretroviral therapy HIVre-emergence generally follows. Thereafter, patients often experiencerapid destruction of HIV-specific CD4 T cells, followed by return toprogression of disease despite prior genetic therapy. By employingselective enrichment for HIV-specific T cells in accordance with thecompositions and methods described herein, a new HIV treatment regimenhas been developed including, in various embodiments, a functional cure.

Definitions and Interpretation

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclature used in connection with, and techniques of, cell and tissueculture, molecular biology, immunology, microbiology, genetics andprotein and nucleic acid chemistry and hybridization described hereinare those well-known and commonly used in the art. The methods andtechniques of the present disclosure are generally performed accordingto conventional methods well-known in the art and as described invarious general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. See, e.g.: Sambrook J. & Russell D. Molecular Cloning: ALaboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (2000); Ausubel et al., Short Protocols in MolecularBiology: A Compendium of Methods from Current Protocols in MolecularBiology, Wiley, John & Sons, Inc. (2002); Harlow and Lane, UsingAntibodies: A Laboratory Manual; Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1998); and Coligan et al., Short Protocols inProtein Science, Wiley, John & Sons, Inc. (2003). Any enzymaticreactions or purification techniques are performed according tomanufacturer's specifications, as commonly accomplished in the art or asdescribed herein. The nomenclature used in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those well-known and commonly used in the art.

As used herein, the term “about” will be understood by persons ofordinary skill in the art and will vary to some extent depending uponthe context in which it is used. If there are uses of the term which arenot clear to persons of ordinary skill in the art given the context inwhich it is used, “about” will mean up to plus or minus 10% of theparticular term.

As used herein, the terms “administration of” or “administering” anactive agent means providing an active agent of the invention to thesubject in need of treatment in a form that can be introduced into thatindividual's body in a therapeutically useful form and therapeuticallyeffective amount.

As used herein, the term “AGT103” refers to a particular embodiment of alentiviral vector that contains a miR30-CCR5/miR21-Vif/miR185-TatmicroRNA cluster sequence, as detailed herein.

As used herein, the term “AGT103T” refers to a cell that has beentransduced with a lentivirus that contains the AGT103 lentiviral vector.

As used herein, the term “CCR5” refers to C-C chemokine receptor 5.Reference herein to “CCR5delta32” is reference to a mutant genotype inthe CCR5 gene.

As used herein, the term “CD” refers to a particular cluster ofdifferentiation protein. A non-limiting example of this terminology asused herein is CD4 protein expression. Examples of such proteinsinclude, but are not limited to CD4.

As used herein, the term “cART” refers to combination antiretroviraltherapy. The term “cART” may be used synonymously with HAART (HighlyActive Antiretroviral Therapy).

Throughout this specification and claims, the word “comprise,” orvariations such as “comprises” or “comprising,” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers. Further, as usedherein, the term “includes” means includes without limitation.

As used herein, the term “engraftment” refers to the ability for oneskilled in the art to determine a quantitative level of sustainedengraftment in a subject following infusion of a cellular source (seefor e.g.: Rosenberg et al., N Engl. J. Med. 323:570-578 (1990); Dudleyel al., J. Immunother. 24:363-373 (2001); Yee et al., Curr. Opin.Immunol. 13:141-146 (2001); Rooney et al., Blood 92:1549-1555 (1998)).

The terms, “expression,” “expressed,” or “encodes” refer to the processby which polynucleotides are transcribed into mRNA and/or the process bywhich the transcribed mRNA is subsequently being translated intopeptides, polypeptides, or proteins. Expression may include splicing ofthe mRNA in a eukaryotic cell or other forms of post-transcriptionalmodification or post-translational modification.

The term “functional cure”, as referenced above, and further definedherein, refers to a state or condition wherein HIV+ individuals whopreviously required ongoing HIV therapies such as cART or HAART, maysurvive with low or undetectable virus replication using lower doses,intermittent doses, alternate drug combinations or single agents, ordiscontinued dosing of such HIV therapies. An individual may be said tohave been “functionally cured” while still requiring adjunct therapy tomaintain low level virus replication and slow or eliminate diseaseprogression. A possible outcome of a functional cure is the eventualeradication of all or virtually all HIV such that no recurrence isdetected within a specified time frame, for example, 1 month, 3 months,6 months, 1 year, 3 years, and 5 years, and all other time frames as maybe defined.

The term “HIV vaccine” encompasses immunogens plus vehicle plus adjuvantintended to elicit HIV-specific immune responses. The term “HIV vaccine”is within the meaning of the term “stimulatory agent” as describedherein. A “HIV vaccine” may include purified or whole inactivated virusparticles that may be HIV or recombinant virus vectors capable ofexpressing HIV proteins, protein fragments or peptides, glycoproteinfragments or glycopeptides, in addition to recombinant bacterialvectors, plasmid DNA or RNA capable of directing cells to producing HIVproteins, glycoproteins or protein fragments able to elicit specificimmunity. Alternately, specific methods for immune stimulation includinganti-CD3/CD28 beads, T cell receptor-specific antibodies, mitogens,superantigens, cytokines and other chemical or biological stimuli may beused to activate dendritic, T or B cells for the purposes of enrichingHIV-specific CD4 T cells prior to transduction or for in vitro assay oflentivirus-transduced CD4 T cells. Activating substances may be soluble,polymeric assemblies, liposome or endosome-based or linked to beads.Cytokines including interleukin-2, 6, 7, 12, 15, 23 or others may beadded to improve cellular responses to stimuli and/or improve thesurvival of CD4 T cells throughout the culture and transductionintervals. Alternately, and without limiting any of the foregoing, theterm “HIV vaccine” encompasses the MVA/HIV62B vaccine and variantsthereof. The MVA/HIV62B vaccine is a known highly attenuated doublerecombinant MVA vaccine. The MVA/HIV62B vaccine was constructed throughthe insertion of HIV-1 gag-pol and env sequences into the known MVAvector (see: for e.g.: Goepfert et al. (2014) J. Infect. Dis. 210(1):99-110, and see WO2006026667, both of which are incorporated herein byreference). The term “HIV vaccine” also includes any one or morevaccines provided in Table 1, below and in any similar tables containedin the priority documents (all of which are incorporated herein in theirentirety).

TABLE 1 IAVI Clinical Trial ID* Prime** HVTN 704 AMP VRC-HIVMAB060-00-ABVAC89220HPX2004 Ad26.Mos.HIV Trivalent 01-I-0079 VRC4302 04/400-003-04APL 400-003 GENEVAX-HIV 10-1074 10-1074 87 I-114 gp160 Vaccine(Immuno-AG) ACTG 326; PACTG 326 ALVAC vCP1452 Ad26.ENVA.01 Ad26.EnvA-01Ad5HVR48.ENVA.01 Ad5HVR48.ENVA.01 ANRS VAC 02 rgp 160 + peptide V3 ANRSVAC 02 ANRS VAC 04 LIPO-6 ANRS VAC 05 ALVAC vCP125 ANRS VAC 07 ALVACvCP300 ANRS VAC 08 ALVAC-HIV MN120TMG strain (vCP205) ANRS VAC 09 bisLIPO-6 ANRS VAC 12 LPHIV1 ANRS VAC 14 gp160 MN/LAI ANRS VAC 16 LPHIV1ANRS VAC 18 LIPO-5 APL 400-003RX101 APL 400-003 GENEVAX-HIV AVEG 002HIVAC-1e AVEG 003 VaxSyn gp160 Vaccine (MicroGeneSys) AVEG 004 gp160Vaccine (Immuno-AG) AVEG 005A/B Env 2-3 AVEG 006X; VEU 006 MN rgp120AVEG 007A/B rgp120/HIV-1 SF-2 AVEG 011 UBI HIV-1 Peptide Immunogen,Multivalent AVEG 013A gp160 Vaccine (Immuno-AG) AVEG 014A/B TBC-3B AVEG017 UBI HIV-1 Peptide Vaccine, Microparticulate Monovalent AVEG 019p17/p24:Ty-VLP AVEG 020 gp120 C4-V3 AVEG 021 P3C541b Lipopeptide AVEG022 ALVAC-HIV MN120TMG strain (vCP205) AVEG 028 Salmonella typhi CVD908-HIV-1 LAI gp 120 AVEG 031 APL 400-047 AVEG 034/034A ALVAC vCP1433C060301 GTU-MultiHIV C86P1 HIV gp140 ZM96 Cervico-vaginal CN54gpCN54gp140 140-hsp70 Conjugate Vaccine (TL01) CM235 and SF2gp120 CM235(ThaiE) gp120 plus SF2(B) gp120 CombiHIVvac CombiHIVvac (KombiVIChvak)CRC282 P2G12 CUTHIVAC002 DNA-C CN54ENV DCVax-001 DCVax-001 DNA-4 DNA-4DP6?001 DP6?001 DNA DVP-1 EnvDNA EN41-UGR7C EN41-UGR7C EnvPro EnvProEuroNeut41 EN41-FPA2 EV01 NYVAC-C EV02 (EuroVacc 02) DNA-C ExtentionHVTN Sub C gp140 073E/SAAVI 102 F4/AS01 F4/AS01 FIT Biotech GTU-NefGuangxi CDC DNA Chinese DNA vaccine HGP-30 memory HGP-30 responsesHIV-CORE002 ChAdV63.HIVconsv HIV-POL-001 MVA-mBN32 HIVIS 01 HIVIS-DNAHIVIS 02 MVA-CMDR HVRF-380-131004 Vichrepol HVTN 040 AVX101 HVTN 041rgp120w61d HVTN 044 VRC-HIVDNA009-00-VP HVTN 045 pGA2/JS7 DNA HVTN 048EP HIV-1090 HVTN 049 Gag and Env DNA/PLG microparticles HVTN 050/Merck018 MRKAd5 HIV-1 gag HVTN 052 VRC-HIVDNA009-00-VP HVTN 054VRC-HIVADV014-00-VP HVTN 055 TBC-M335 HVTN 056 MEP HVTN 059 AVX101 HVTN060 HIV-1 gag DNA HVTN 064 EP HIV-1043 HVTN 065 pGA2/JS7 DNA HVTN 067EP-1233 HVTN 070 PENNVAX-B HVTN 072 VRC-HIVDNA044-00-VP HVTN 073 SAAVIDNA-C2 HVTN 076 VRC-HIVDNA016-00-VP HVTN 077 VRC-HIVADV027-00-VP HVTN078 NYVAC-B HVTN 082 VRC-HIVDNA016-00-VP HVTN 084 VRC-HIVADV054-00-VPHVTN 086, SAAVI 103 SAAVI MVA-C HVTN 087 HIV-MAG HVTN 088 Oligomericgp140/MF59 HVTN 090 VSV-Indiana HIV gag vaccine HVTN 092 DNA-HIV-PT123HVTN 094 GEO-D03 HVTN 096 DNA-HIV-PT123 HVTN 097 ALVAC-HIV vCP1521 HVTN100 ALVAC-HIV-C (vCP2438) HVTN 101 DNA-HIV-PT123 HVTN 104VRC-HIVMAB060-00-AB HVTN 105 AIDSVAX B/E HVTN 106 DNA Nat-B env HVTN 110Ad4-mgag HVTN 112 HIV-1 nef/tat/vif, env pDNA vaccine HVTN 116VRC-HIVMAB060-00-AB HVTN 205 pGA2/JS7 DNA HVTN 702 ALVAC-HIV-C (vCP2438)HVTN 703 AMP VRC-HIVMAB060-00-AB HVTN 908 pGA2/JS7 DNA IAVI 001 DNA.HIVAIAVI 016 MVA.HIVA IAVI A001 tgAAC09 IAVI A003 AAV1-PG9 IAVI B001Ad35-GRIN/ENV IAVI B002 Adjuvanted GSK investigational HIV vaccineformulation 1 IAVI B003 Ad26.EnvA-01 IAVI B004 HIV-MAG IAVI C001 ADVAXIAVI C002 ADMVA IAVI D001 TBC-M4 IAVI N004 Ad35-GRIN HIV-CORE 004 IAVIR001 rcAd26.MOS1.HIVEnv IAVI S001 SeV-G IDEA EV06 DNA-HIV-PT123 IHV01Full-Length Single Chain (FLSC) IMPAACT P1112 VRC-HIVMAB060-00-ABIPCAVD006 MVA mosaic IPCAVD008 Trimeric gp140 IPCAVD009 Ad26.Mos.HIVTrivalent ISS P-001 Tat vaccine LFn-p24 vaccine LFn-p24 MCA-0835 3BNC117Mucovac2 CN54gp140 MV1-F4 Measles Vector - GSK MYM-V101 Virosome-Gp41NCHECR-AE1 pHIS-HIV-AE PEACHI-04 ChAdV63.HIVconsv PedVacc001 & MVA.HIVAPedVacc002 PolyEnv1 PolyEnv1 PXVX-HIV-100-001 Ad4-mgag RISVAC02 MVA-B RV151/WRAIR 984 LFn-p24 RV 158 MVA-CMDR SG06RS02 HIV gp140 ZM96 TAB9 TAB9TaMoVac II HIVIS-DNA UBI V106 UBI HIV-1 Peptide Vaccine,Microparticulate Monovalent UCLA MIG-001 TBC-3B UKHVCSpoke003 DNA -CN54ENV and ZM96GPN V3-MAPS V3-MAPS VAX 002 AIDSVAX B/B VAX 003 AIDSVAXB/E VRC 602 VRC-HIVMAB060-00-AB VRC 607 VRCHIVMAB080-00-AB *IAVI is theInternational AIDS Vaccine Initiative, whose clinical trials database ispublicly available at http://www.iavi.org/trials-database/trials. **Asused herein, the term “Prime” refers to the composition initially usedas an immunological inoculant in a given clinical trial as referenced inTable 1 herein.

The term “in vivo” refers to processes that occur in a living organism.The term “ex vivo” refers to processes that occur outside of a livingorganism. For example, in vivo treatment refers to treatment that occurswithin a patient's body, while ex vivo treatment is one that occursoutside of a patient's body, but still uses or accesses or interactswith tissues from that patient. Thereafter, an ex vivo treatment stepmay include a subsequent in vivo treatment step.

The term “miRNA” refers to a microRNA, and also may be referred toherein as “miR”. The term “microRNA cluster” refers to at least twomicroRNAs that are situate on a vector in close proximity to each otherand are co-expressed.

The term “packaging cell line” refers to any cell line that can be usedto express a lentiviral particle.

The term “PBMC” refers to peripheral blood mononuclear cells.

The term “percent identity,” in the context of two or more nucleic acidor polypeptide sequences, refer to two or more sequences or subsequencesthat have a specified percentage of nucleotides or amino acid residuesthat are the same, when compared and aligned for maximum correspondence,as measured using one of the sequence comparison algorithms describedbelow (e.g., BLASTP and BLASTN or other algorithms available to personsof ordinary skill in the art) or by visual inspection. Depending on theapplication, the “percent identity” can exist over a region of thesequence being compared, e.g., over a functional domain, or,alternatively, exist over the full length of the two sequences to becompared. For sequence comparison, typically one sequence acts as areference sequence to which test sequences are compared. When using asequence comparison algorithm, test and reference sequences are inputinto a computer, subsequence coordinates are designated, if necessary,and sequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information website.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Thepercent identity between two nucleotide or amino acid sequences can alsobe determined using the algorithm of E. Meyers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences of the present disclosure canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify related sequences. Such searches canbe performed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acidmolecules of the invention. BLAST protein searches can be performed withthe XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to the protein molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., (BLAST and NBLAST)can be used. See http://www.ncbi.nlm.nih.gov.

As used herein, “pharmaceutically acceptable” refers to those compounds,materials, compositions, and/or dosage forms which are, within the scopeof sound medical judgment, suitable for use in contact with the tissues,organs, and/or bodily fluids of human beings and animals withoutexcessive toxicity, irritation, allergic response, or other problems orcomplications commensurate with a reasonable benefit/risk ratio.

As used herein, a “pharmaceutically acceptable carrier” refers to, andincludes, any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible. Thecompositions can include a pharmaceutically acceptable salt, e.g., anacid addition salt or a base addition salt (see, e.g., Berge et al.(1977) J Pharm Sci 66:1-19).

As used herein, the term “physical method of selection” refers to anyphysical method that can be used to positively select for a cell typewithin a larger mixture of cells (e.g., PBMC). A non-limiting example ofa physical method of selection is magnetic bead sorting.

As used herein, the term “SEQ ID NO” is synonymous with the term“Sequence ID No.”

As used herein, “small RNA” refers to non-coding RNA that are generallyless than about 200 nucleotides or less in length and possess asilencing or interference function. In other embodiments, the small RNAis about 175 nucleotides or less, about 150 nucleotides or less, about125 nucleotides or less, about 100 nucleotides or less, or about 75nucleotides or less in length. Such RNAs include microRNA (miRNA), smallinterfering RNA (siRNA), double stranded RNA (dsRNA), and short hairpinRNA (shRNA). “Small RNA” of the disclosure should be capable ofinhibiting or knocking-down gene expression of a target gene, forexample through pathways that result in the destruction of the targetgene mRNA.

As used herein, the term “stimulatory agent” refers to any exogenousagent that can stimulate an immune response, and includes, withoutlimitation, a vaccine, a HIV vaccine, and HIV or HIV-related peptides. Astimulatory agent can preferably stimulate a T cell response.

As used herein, the term “subject” includes a human patient but alsoincludes other mammals. The terms “subject,” “individual,” “host,” and“patient” may be used interchangeably herein.

The term “Tat” refers to the HIV tat gene and its gene product, andvariants thereof.

The term “therapeutically effective amount” refers to a sufficientquantity of the active agents of the present invention, in a suitablecomposition, and in a suitable dosage form to treat or prevent thesymptoms, progression, or onset of the complications seen in patientssuffering from a given ailment, injury, disease, or condition. Thetherapeutically effective amount will vary depending on the state of thepatient's condition or its severity, and the age, weight, etc., of thesubject to be treated. A therapeutically effective amount can vary,depending on any of a number of factors, including, e.g., the route ofadministration, the condition of the subject, as well as other factorsunderstood by those in the art.

As used herein, the term “therapeutic vector” is synonymous with alentiviral vector such as the AGT103 vector.

The term “treatment” or “treating” generally refers to an interventionin an attempt to alter the natural course of the subject being treated,and can be performed either for prophylaxis or during the course ofclinical pathology. Desirable effects include, but are not limited to,preventing occurrence or recurrence of disease, alleviating symptoms,suppressing, diminishing or inhibiting any direct or indirectpathological consequences of the disease, ameliorating or palliating thedisease state, and causing remission or improved prognosis.

The term “vaccine”, which is used interchangeably with the term“therapeutic vaccine” refers to an exogenous agent that can elicit animmune response in an individual and includes, without limitation,purified proteins, inactivated viruses, virally vectored proteins,bacterially vectored proteins, peptides or peptide fragments, orvirus-like particles (VLPs).

The term “Vif” refers to the HIV vif gene and its gene product, andvariants thereof.

Description of Aspects of the Disclosure

As detailed herein, in one aspect, a method of treating cells infectedwith HIV is provided. The method generally includes contactingperipheral blood mononuclear cells (PBMC) isolated from a subjectinfected with HIV with a therapeutically effective amount of astimulatory agent, wherein the contacting step is carried out ex vivo;transducing the PBMC ex vivo with a viral delivery system encoding atleast one genetic element; and culturing the transduced PBMC for aperiod of time sufficient to achieve such transduction. In embodiments,the transduced PBMC are cultured from about 1 to about 35 days. Inembodiments, the method further includes infusing the transduced PBMCinto a subject. In embodiments, the subject is a human. In embodiments,the stimulatory agent is a peptide or mixture of peptides, and inembodiments includes a gag peptide. In further embodiments, thestimulatory includes a vaccine. In embodiments, the vaccine is a HIVvaccine, and in further embodiments, the HIV vaccine is a MVA/HIV62Bvaccine or a variant thereof. In embodiments, the viral delivery systemincludes a lentiviral particle. In embodiments, the at least one geneticelement includes a small RNA capable of inhibiting production ofchemokine receptor CCR5. In embodiments, the at least one geneticelement includes at least one small RNA capable of targeting an HIV RNAsequence. In other embodiments, the at least one genetic elementincludes a small RNA capable of inhibiting production of chemokinereceptor CCR5 and at least one small RNA capable of targeting an HIV RNAsequence. In embodiments, the HIV RNA sequence includes a HIV Vifsequence, a HIV Tat sequence, or variants thereof. In embodiments, theat least one genetic element includes at least one of a microRNA or ashRNA. In further embodiments, the at least one genetic elementcomprises a microRNA cluster.

In another aspect, a method is disclosed which includes obtainingperipheral blood from HIV+ individuals; fractionating the blood toobtain a PBMC population; contacting the PBMC population with purifiedantigen-presenting cells or peptides or proteins representing componentsof HIV; culturing the contacted PBMC population for about 1 to about 12days to expand an antigen-specific population; positively selectingcells that respond to peptide stimulation to produce an enriched cellfraction; transducing the enriched cell fraction ex vivo with a viraldelivery system as detailed herein, and culturing the transduced cellfraction for a period of time sufficient to ensure adequatetransduction.

In embodiments, the PBMC population is further purified to produce apurified fraction of PBMC. In embodiments, further purified fractions ofPBMC are contacted with peptides or proteins representing components ofHIV.

In another aspect, the at least one genetic element includes a microRNAhaving at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95% or more percent identity with SEQ ID NO: 1. Inembodiments, the at least one genetic element comprises SEQ ID NO: 1.

In another aspect, the at least one genetic element includes a microRNAhaving at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95% or more percent identity with SEQ ID NO: 2; or atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95% or more percent identity with SEQ ID NO: 3. In embodiments,the at least one genetic element includes SEQ ID NO: 2 or SEQ ID NO: 3.

In another aspect, the microRNA cluster includes a sequence having atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95% or more percent identity with SEQ ID NO: 31. In embodiments,the microRNA cluster includes SEQ ID NO: 31.

In another aspect, a method of manufacturing a cell product for treatingHIV infection in a subject is disclosed. The method generally includesobtaining blood leukocytes; removing leukocytes from the subject andpurifying peripheral blood mononuclear cells (PBMC) or defined fractionsof PBMC. The method further includes contacting the PBMC or purifiedfraction of PBMC ex vivo with a therapeutically effective amount of astimulatory agent; positive selection based on response to stimulatoryagent to enrich the proportion of antigen-specific T cells; transducingthe PBMC or purified fraction of PBMC ex vivo with a viral deliverysystem encoding at least one genetic element; and culturing thetransduced PBMC or a purified fraction of PBMC for a period of timesufficient to achieve transduction and growth of the modified cellpopulation. The method may further include further enrichment of thePBMC, for example, by preferably enriching the PBMC for CD4+ T cells orselecting for antigen-specific cells based on cytokine expression orcombinations of selection methods to enrich for the therapeutic fractionof cells. In embodiments, the transduced PBMC or purified fraction ofPBMC are cultured from about 1 to about 35 days. The method may furtherinvolve infusing the transduced PBMC or purified fraction of PBMC into asubject. The subject may be a human. The at least one of the firststimulatory agents may include a peptide or mixture of peptides and mayrepresent one, two, three or more of proteins encoded by the HIV genome.In embodiments, at least one of the first stimulatory agents includes agag peptide. The at least one of the first stimulatory agents mayinclude a vaccine. In embodiments, the vaccine is a HIV vaccine, and infurther embodiments, the HIV vaccine is a MVA/HIV62B vaccine or avariant thereof. In embodiments, the first stimulatory agent is amixture of gag peptides.

In embodiments, the viral delivery system includes a lentiviralparticle. In embodiments, the at least one genetic element includes asmall RNA capable of inhibiting production of chemokine receptor CCR5.In embodiments, the at least one genetic element includes at least onesmall RNA capable of targeting an HIV RNA sequence. In embodiments, theat least one genetic element includes a small RNA capable of inhibitingproduction of chemokine receptor CCR5 and at least one small RNA capableof targeting an HIV RNA sequence. The HIV RNA sequence may include a HIVVif sequence, a HIV Tat sequence, or variants thereof. The at least onegenetic element may include a microRNA or a shRNA, or a cluster thereof.In embodiments, the at least one genetic element comprises a syntheticmicroRNA cluster.

In another aspect, the at least one genetic element includes a microRNAhaving at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95% or more percent identity with SEQ ID NO: 1. Inembodiments, the at least one genetic element comprises SEQ ID NO: 1.

In another aspect, the at least one genetic element includes a microRNAhaving at least 80%, or at least 85%, or at least 90%, or at least 95%percent identity with SEQ ID NO: 2; or at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% or more percentidentity with SEQ ID NO: 3. In embodiments, the at least one geneticelement includes SEQ ID NO: 2 or SEQ ID NO: 3.

In another aspect, the microRNA cluster includes a sequence having atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95% or more percent identity with SEQ ID NO: 31. In embodiments,the microRNA cluster includes SEQ ID NO: 31.

In another aspect, a lentiviral vector is disclosed. The lentiviralvector includes at least one encoded genetic element, wherein the atleast one encoded genetic element comprises a small RNA capable ofinhibiting production of chemokine receptor CCR5 or at least one smallRNA capable of targeting an HIV RNA sequence. In another aspect alentiviral vector is disclosed in the at least one encoded geneticelement comprises a small RNA capable of inhibiting production ofchemokine receptor CCR5 and at least one small RNA capable of targetingan HIV RNA sequence. The HIV RNA sequence may include a HIV Vifsequence, a HIV Tat sequence, or a variant thereof. The at least oneencoded genetic element may include a microRNA or a shRNA. The at leastone encoded genetic element may include a microRNA cluster.

In another aspect, the at least one genetic element includes a microRNAhaving at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95% or more percent identity with SEQ ID NO: 1. Inembodiments, the at least one genetic element comprises SEQ ID NO: 1.

In another aspect, the at least one genetic element includes a microRNAhaving at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95% or more percent identity with SEQ ID NO: 2; or atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95% or more percent identity with SEQ ID NO: 3. In embodiments,the at least one genetic element includes SEQ ID NO: 2; or SEQ ID NO: 3.

In another aspect, the microRNA cluster includes a sequence having atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95% or more percent identity with SEQ ID NO: 31. In embodiments,the microRNA cluster includes SEQ ID NO: 31.

In another aspect, a lentiviral vector system for expressing alentiviral particle is provided. The system includes a lentiviral vectoras described herein; at least one envelope plasmid for expressing anenvelope protein preferably optimized for infecting a cell; and at leastone helper plasmid for expressing a gene of interest, for example any ofgag, pol, and rev genes, wherein when the lentiviral vector, the atleast one envelope plasmid, and the at least one helper plasmid aretransfected into a packaging cell, wherein a lentiviral particle isproduced by the packaging cell, wherein the lentiviral particle iscapable of modulating a target sequence of interest, for exampleinhibiting production of chemokine receptor CCR5 or targeting an HIV RNAsequence.

In another aspect, a lentiviral particle capable of infecting a cell isdisclosed. The lentiviral particle includes at least one envelopeprotein preferably optimized for infecting a cell, and a lentiviralvector as described herein. The envelope protein may be optimized forinfecting a T cell. In embodiments, the envelope protein is optimizedfor infecting a CD4+ T cell.

In another aspect, a modified cell is disclosed. In embodiments, themodified cell is a CD4+ T cell. In embodiments, the CD4+ T cell isinfected with a lentiviral particle as described herein. In embodiments,the CD4+ T cell also has been selected to recognize an HIV antigen basedon the prior immunization with a stimulatory agent. In a furtherembodiment, the HIV antigen that is recognized by the CD4+ T cellincludes a gag antigen. In a further embodiment, the CD4+ T cellexpresses a decreased level of CCR5 following infection with thelentiviral particle.

In another aspect, a method of selecting a subject for a therapeutictreatment regimen is disclosed. The method generally includes immunizingthe subject with an effective amount of a first stimulatory agent;removing leukocytes from the subject and purifying peripheral bloodmononuclear cells (PBMC) and determining a first quantifiablemeasurement associated with at least one factor associated with thePBMC; contacting the PBMC ex vivo with a therapeutically effectiveamount of a second stimulatory agent, and determining a secondmeasurement associated with the at least one factor associated with thePBMC, whereby when the second quantifiable measurement is different(e.g., higher) than the first quantifiable measurement, the subject isselected for the treatment regimen. The at least one factor may be Tcell proliferation or IFN gamma production.

Human Immunodeficiency Virus (HIV)

Human Immunodeficiency Virus, which is also commonly referred to as“HIV”, is a retrovirus that causes acquired immunodeficiency syndrome(AIDS) in humans. AIDS is a condition in which progressive failure ofthe immune system allows life-threatening opportunistic infections andcancers to thrive. Without treatment, average survival time afterinfection with HIV is estimated to be 9 to 11 years, depending upon theHIV subtype and genetics of the host population. Infection with HIVoccurs by the transfer of bodily fluids, including but not limited toblood, semen, vaginal fluid, pre-ejaculate, saliva, tears, lymph orcerebro-spinal fluid, or breast milk, or use of contaminated blood ortissue products. HIV may be present in an infected individual as bothfree virus particles and within infected immune cells.

HIV infects vital cells in the human immune system such as helper Tcells, although tropism can vary among HIV subtypes. Immune cells thatmay be specifically susceptible to HIV infection include but are notlimited to CD4+ T cells, macrophages, and dendritic cells. HIV infectionleads to low levels of CD4+ T cells through a number of mechanisms,including but not limited to apoptosis of uninfected bystander cells,direct viral killing of infected cells, and killing of infected CD4+ Tcells by CD8 cytotoxic lymphocytes that recognize infected cells. WhenCD4+ T cell numbers decline below a critical level, cell-mediatedimmunity is lost, and the body becomes progressively more susceptible toopportunistic infections and cancer.

Structurally, HIV is distinct from many other retroviruses. The RNAgenome consists of at least seven structural landmarks (LTR, TAR, RRE,PE, SLIP, CRS, and INS), and at least nine genes (gag, pol, env, tat,rev, nef, vif, vpr, vpu, and sometimes a tenth tev, which is a fusion oftat, env and rev), encoding 19 proteins. Three of these genes, gag, pol,and env, contain information needed to make the structural proteins fornew virus particles.

HIV replicates primarily in CD4 T cells, and causes cellular destructionor dysregulation to reduce host immunity. Because HIV establishesinfection as an integrated provirus and may enter a state of latencywherein virus expression in a particular cell decreases below the levelfor cytopathology affecting that cell or detection by the host immunesystem, HIV is difficult to treat and has not been eradicated even afterprolonged intervals of combination antiretroviral therapy (cART). Inmost cases, HIV infection causes fatal disease although survival may beprolonged by cART.

A major goal in the fight against HIV is to develop strategies forcuring disease. Prolonged cART has not accomplished this goal, soinvestigators have turned to alternative procedures. Early efforts toimprove host immunity by therapeutic immunization (using a vaccine afterinfection has occurred) had marginal or no impact. Likewise, treatmentintensification had moderate or no impact.

Some progress has been made using genetic therapy, but positive resultsare sporadic and found only among rare human beings carrying defects inone or both alleles of the gene encoding CCR5, which plays a criticalrole in viral penetration of host cells. However, many investigators areoptimistic that genetic therapy holds the best promise for eventuallyachieving an HIV cure.

As disclosed herein, the methods and compositions of the invention areable to achieve a functional cure that may or may not include completeeradication of all HIV from the body. As mentioned above, a functionalcure is defined as a state or condition wherein HIV+ individuals whopreviously required cART, may survive with low or undetectable virusreplication and using lower or intermittent doses of cART, or arepotentially able to discontinue cART altogether. As used herein, afunctional cure may still possibly require adjunct therapy to maintainlow level virus replication and slow or eliminate disease progression. Apossible outcome of a functional cure is the eventual eradication of HIVto prevent all possibility of recurrence.

The primary obstacles to achieving a functional cure lie in the basicbiology of HIV itself. Virus infection deletes CD4 T cells that arecritical for nearly all immune functions. Most importantly, HIVinfection and depletion of CD4 T cells requires activation of individualcells. Activation is a specific mechanism for individual CD4 T cellclones that recognize pathogens or other molecules, using a rearranged Tcell receptor.

In the case of HIV, infection activates a population of HIV-specific Tcells that become infected and are consequently depleted before other Tcells that are less specific for the virus, which effectively cripplesthe immune system's defense against the virus. The capacity forHIV-specific T cell responses is rebuilt during prolonged cART; however,when cART is interrupted the rebounding virus infection repeats theprocess and again deletes the virus-specific cells, resetting the clockon disease progression.

Clearly, a functional cure is only possible if enough HIV-specific CD4 Tcells are protected to allow for a host's native immunity to confrontand control HIV once cART is interrupted. In one embodiment, the presentinvention provides methods and compositions for improving theeffectiveness of genetic therapy to provide a functional cure of HIVdisease. In another embodiment, the present invention provides methodsand compositions for enhancing host immunity against HIV to provide afunctional cure. In yet another embodiment, the present inventionprovides methods and compositions for enriching HIV-specific CD4 T cellsin a patient to achieve a functional cure.

In one embodiment of the invention, treatment results in enriching asubject's HIV-specific CD4 T cells by about 100%, about 200%, about300%, about 400%, about 500%, about 600%, about 700%, about 800%, about900%, or about 1000%.

Gene Therapy

Viral vectors are used to deliver genetic constructs to host cells forthe purposes of disease therapy or prevention.

Genetic constructs can include, but are not limited to, functional genesor portions of genes to correct or complement existing defects, DNAsequences encoding regulatory proteins, DNA sequences encodingregulatory RNA molecules including antisense, short homology RNA, longnon-coding RNA, small interfering RNA or others, and decoy sequencesencoding either RNA or proteins designed to compete for criticalcellular factors to alter a disease state. Gene therapy involvesdelivering these therapeutic genetic constructs to target cells toprovide treatment or alleviation of a particular disease.

There are multiple ongoing efforts to utilize genetic therapy in thetreatment of HIV disease, but thus far, the results have been poor. Asmall number of treatment successes were obtained in rare HIV patientscarrying a spontaneous deletion of the CCR5 gene (an allele known asCCR5delta32).

Lentivirus-delivered nucleases or other mechanisms for genedeletion/modification may be used to lower the overall expression ofCCR5 and/or help to lower HIV replication. At least one study hasreported having success in treating the disease when lentivirus wasadministered in patients with a genetic background of CCR5delta32.However, this was only one example of success, and many other patientswithout the CCR5delta32 genotype have not been treated as successfully.Consequently, there is a substantial need to improve the performance ofviral genetic therapy against HIV, both in terms of performance for theindividual viral vector construct and for improved use of the vectorthrough a strategy for achieving functional HIV cure.

For example, some existing therapies rely on zinc finger nucleases todelete a portion of CCR5 in an attempt to render cells resistant to HIVinfection. However, even after optimal treatment, only 30% of T cellshad been modified by the nuclease at all, and of those that weremodified, only 10% of the total CD4 T cell population had been modifiedin a way that would prevent HIV infection. In contrast, the disclosedmethods result in virtually every cell carrying a lentivirus transgenehaving a reduction in CCR5 expression below the level needed to allowHIV infection.

For the purposes of the disclosed methods, gene therapy can include, butis not limited to, affinity-enhanced T cell receptors, chimeric antigenreceptors on CD4 T cells (or alternatively on CD8 T cells), modificationof signal transduction pathways to avoid cell death cause by viralproteins, increased expression of HIV restriction elements includingTREX, SAMHD1, MxA or MxB proteins, APOBEC complexes, TRIMS-alphacomplexes, tetherin (BST2), and similar proteins identified as beingcapable of reducing HIV replication in mammalian cells.

Immunotherapy

Historically, vaccines have been a go-to weapon against deadlyinfectious diseases, including smallpox, polio, measles, and yellowfever. Unfortunately, there is no currently approved vaccine for HIV.The HIV virus has unique ways of evading the immune system, and thehuman body seems incapable of mounting an effective immune responseagainst it. As a result, scientists do not have a clear picture of whatis needed to provide protection against HIV.

However, immunotherapy may provide a solution that was previouslyunaddressed by conventional vaccine approaches. Immunotherapy, alsocalled biologic therapy, is a type of treatment designed to boost thebody's natural defenses to fight infections or cancer. It uses materialseither made by the body or in a laboratory to improve, target, orrestore immune system function.

In some embodiments of the disclosed invention, immunotherapeuticapproaches may be used to enrich a population of HIV-specific CD4 Tcells for the purpose of increasing the host's anti-HIV immunity. Insome embodiments of the disclosed invention, integrating ornon-integrating lentivirus vectors may be used to transduce a host'simmune cells for the purposes of increasing the host's anti-HIVimmunity. In yet another embodiment of the invention, a vaccinecomprising HIV proteins including but not limited to a killed particle,a virus-like particle, HIV peptides or peptide fragments, a recombinantviral vector, a recombinant bacterial vector, a purified subunit orplasmid DNA combined with a suitable vehicle and/or biological orchemical adjuvants to increase a host's immune responses may be used toenrich the population of virus-specific T cells or antibodies, and thesemethods may be further enhanced through the use of HIV-targeted genetictherapy using lentivirus or other viral vector.

Methods

In one aspect, the disclosure provides methods for using viral vectorsto achieve a functional cure for HIV disease. The methods generallyinclude immunotherapy to enrich the proportion of HIV-specific CD4 Tcells, followed by lentivirus transduction to deliver inhibitors of HIVand CCR5 and CXCR4 as required.

In one embodiment, the methods include a first stimulation event toenrich a proportion of HIV-specific CD4 T cells. The first stimulationcan include administration of one or more of any agent suitable forenriching a patient's HIV-specific CD4+ T cells including but notlimited to a vaccine.

Therapeutic vaccines can include one or more HIV protein with proteinsequences representing the predominant viral types of the geographicregion where treatment is occurring. Therapeutic vaccines will includepurified proteins, inactivated viruses, virally vectored proteins,bacterially vectored proteins, peptides or peptide fragments, virus-likeparticles (VLPs), biological or chemical adjuvants including cytokinesand/or chemokines, vehicles, and methods for immunization. Vaccinationsmay be administered according to standard methods known in the art andHIV patients may continue antiretroviral therapy during the interval ofimmunization and subsequent ex vivo lymphocyte culture includinglentivirus transduction.

In some embodiments, HIV+ patients are immunized with an HIV vaccine,increasing the frequency of HIV-specific CD4 T cells by about 2, about25, about 250, about 500, about 750, about 1000, about 1250, or about1500-fold (or any amount in between these values). The vaccine may beany clinically utilized or experimental HIV vaccine, including thedisclosed lentiviral, other viral vectors or other bacterial vectorsused as vaccine delivery systems. In another embodiment, the vectorsencode virus-like particles (VLPs) to induce higher titers ofneutralizing antibodies. In another embodiment, the vectors encodepeptides or peptide fragments associated with HIV including but notlimited to gag, pol, and env, tat, rev, nef, vif, vpr, vpu, and tev, aswell as LTR, TAR, RRE, PE, SLIP, CRS, and INS. Alternatively, the HIVvaccine used in the disclosed methods may comprise purified proteins,inactivated viruses, virally vectored proteins, bacterially vectoredproteins, peptides or peptide fragments, virus-like particles (VLPs), orbiological or chemical adjuvants including cytokines and/or chemokines.

In one embodiment, the methods include ex vivo re-stimulation of CD4 Tcells from persons or patients previously immunized by therapeuticvaccination, using purified proteins, inactivated viruses, virallyvectored proteins, bacterially vectored proteins, biological or chemicaladjuvants including cytokines and/or chemokines, vehicles, and methodsfor re-stimulation. Ex vivo re-stimulation may be performed using thesame vaccine or immune stimulating compound used for in vivoimmunization, or it may be performed using a different vaccine or immunestimulating compound than those used for in vivo immunization. Moreover,in some embodiments, the patient does not require prior therapeuticvaccination or re-stimulation of CD4 T cells if the individual hassufficiently high antigen-specific CD4 T cell responses to HIV proteins.In these embodiments, such a patient may only require ex vivostimulation of CD4 T cells with viral antigens, vaccines or peptidesfollowed by selection for HIV-specific T cells based on the response tostimulation. Enriched cell preparations may include 1%, 5%, 10%, 20%,30%, 40%, 50% or more of the HIV-specific CD4+ T cells and are used forlentivirus transduction of genes able to protection from HIV-mediateddepletion. Stimulation with polyclonal mitogen plus cytokines increasesthe number of enriched and transduced T cells until appropriate levelsare reached for infusion back into the original patient.

In embodiments, peripheral blood mononuclear cells (PBMCs) are obtainedby leukapheresis and treated ex vivo to obtain about 1×10⁹ CD4 T cellsof which about 0.1%, about 1%, about 5% or about 10% or about 30% orabout 40% or about 50% are both HIV-specific in terms of antigenresponses, and HIV-resistant by virtue of carrying the therapeutictransgene delivered by the disclosed lentivirus vector. Alternatively,about 1×10⁷, about 1×10⁸, about 1×10⁹, about 1×10¹⁰, about 1×10¹¹, orabout 1×10¹² CD4 T cells may be isolated for re-stimulation. Anysuitable amount of CD4 T cells are isolated for ex vivo re-stimulation.

The isolated CD4 T cells can be cultured in appropriate mediumthroughout re-stimulation with HIV vaccine antigens, which may includeantigens present in the prior therapeutic vaccination. Antiretroviraltherapeutic drugs including inhibitors of reverse transcriptase,protease or integrase may be added to prevent virus re-emergence duringprolonged ex vivo culture. CD4 T cell re-stimulation is used to enrichthe proportion of HIV-specific CD4 T cells in culture. The sameprocedure may also be used for analytical objectives wherein smallerblood volumes with peripheral blood mononuclear cells obtained bypurification, are used to identify HIV-specific T cells and measure thefrequency of this sub-population.

The PBMC fraction may be enriched for HIV-specific CD4 T cells bycontacting the cells with HIV proteins matching or complementary to thecomponents of the vaccine previously used for in vivo immunization. Exvivo re-stimulation can increase the relative frequency of HIV-specificCD4 T cells by about 2, about 5, about 10, about 25, about 50, about 75,about 100, about 125, about 150, about 175, or about 200-fold. Furtherenrichment is obtained by positive selection for cells responding to HIVantigens, vaccines or peptides. Positive selection is accomplished, forexample, with the CliniMACS Cytokine Capture System (Miltenyi BiotecProduct number 130-028-701, San Diego, Calif. 92121) or similar manualor automated system including the Miltenyi Prodigy System (MiltenyiBiotec Product number 200-075-301, San Diego, Calif. 92121) that iscompatible with selecting viable cells based on expression of a cytokine(including but not limited to interferon gamma or tumor necrosis factoralpha) that is captured by a bi-specific reagent and labeled with amagnetic bead antibody to enable positive selection on a magneticcolumn. Enrichment may also be accomplished by labeling stimulated cellswith antibodies capable of detecting cell surface markers expressedpreferentially on activated T cells including CD45RO, MHC Class II andothers known in the art. Purification of labeled cells may be byfluorescence activated cell sorting, magnetic bead sorting or otherphysical methods capable of purifying viable cells based on phenotypiccharacteristics.

The methods additionally include combining in vivo therapeuticimmunization and ex vivo re-stimulation of CD4 T cells with ex vivolentiviral transduction and culturing.

Thus, in one embodiment, the re-stimulated PBMC or fraction of PBMC thathas been enriched for HIV-specific CD4 T cells can be positivelyselected as described above, cultured for 1, 2, 3, 4, 5 or up to 12, 20or 30 days before activating again with a polyclonal mitogen such asMiltenyi GMP TransAct T cell reagent (Miltenyi Biotec Product number170-076-156, San Diego, Calif. 92121) or other plant- or fungal basedagglutinins or other reagents capable of recognizing cell surface CD3and CD28 to cross link these molecules and cause polyclonal T cellactivation. After polyclonal stimulation cells are transduced withtherapeutic anti-HIV lentivirus or other vectors and maintained inculture for a sufficient period of time for such transduction, forexample from about 1 to about 21 days, including up to about 35 days.Alternatively, the cells may be cultured for about 1-about 18 days,about 1-about 15 days, about 1-about 12 days, about 1-about 9 days, orabout 3-about 7 days. Thus, the transduced cells may be cultured forabout 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 9, about 10, about 11, about 12, about 13, about 14, about 15,about 16, about 17, about 18, about 19, about 20, about 21, about 22,about 23, about 24, about 25, about 26, about 27, about 28, about 29,about 30, about 31, about 32, about 33, about 34, or about 35 days.Activation with a polyclonal mitogen may or may not be included in thecell product manufacturing process.

In further embodiments, once the transduced cells have been cultured fora sufficient period of time, transduced CD4 T cells are infused backinto the original patient. Infusion can be performed using variousdevices and methods known in the art. In some embodiments, infusion maybe accompanied by pre-treatment with cyclophosphamide or similarcompounds to increase the efficiency of re-engraftment.

In some embodiments, a CCR5-targeted therapy may be added to a subject'santiretroviral therapy regimen, which was continued throughout thetreatment process. Examples of CCR5-targeted therapies include but arenot limited to Maraviroc (a CCR5 antagonist) or Rapamycin(immunosuppressive agent that lowers CCR5). In some embodiments, theantiretroviral therapy may be ceased and the subject can be tested forvirus rebound. If no rebound occurs, adjuvant therapy can also beremoved and the subject can be tested again for virus rebound.

In various embodiments, continued virus suppression with reduced or noantiretroviral therapy including cART or HAART, and reduced or noadjuvant therapy for about 26 weeks can be considered a functional curefor HIV. Other definitions of a functional cure are described herein.

The lentiviral and other vectors used in the disclosed methods mayencode at least one, at least two, at least three, at least four, or atleast five genes, or at least six genes, or at least seven genes, or atleast eight genes, or at least nine genes, or at least ten genes, or atleast eleven genes, or at least twelve genes of interest. Given theversatility and therapeutic potential of HIV-targeted gene therapy, aviral vector of the invention may encode genes or nucleic acid sequencesthat include but are not limited to (i) an antibody directed to anantigen associated with an infectious disease or a toxin produced by theinfectious pathogen, (ii) cytokines including interleukins that arerequired for immune cell growth or function and may be therapeutic forimmune dysregulation encountered in HIV and other chronic or acute humanviral or bacterial pathogens, (iii) factors that suppress the growth ofHIV in vivo including CD8 suppressor factors, (iv) mutations ordeletions of chemokine receptor CCR5, mutations or deletions ofchemokine receptor CXCR4, or mutations or deletions of chemokinereceptor CXCR5, (v) antisense DNA or RNA against specific receptors orpeptides associated with HIV or host protein associated with HIV, (vi)small interfering RNA against specific receptors or peptides associatedwith HIV or host protein associated with HIV, or (vii) a variety ofother therapeutically useful sequences that may be used to treat HIV orAIDS.

Additional examples of HIV-targeted gene therapy that can be used in thedisclosed methods include, but are not limited to, affinity-enhanced Tcell receptors, chimeric antigen receptors on CD4 T cells (oralternatively on CD8 T cells), modification of signal transductionpathways to avoid cell death cause by viral proteins, increasedexpression of HIV restriction elements including TREX, SAMHD1, MxA orMxB proteins, APOBEC complexes, TRIMS-alpha complexes, tetherin (BST2),and similar proteins identified as being capable of reducing HIVreplication in mammalian cells.

In some embodiments, a patient may be undergoing cART or HAARTconcurrently while being treated according to the methods of theinvention. In other embodiments, a patient may undergo cART or HAARTbefore or after being treated according to the methods of the invention.In some embodiments, cART or HAART is maintained throughout treatmentaccording to the methods of the invention and the patient may bemonitored for HIV viral burden in blood and frequency oflentivirus-transduced CD4 T cells in blood. Preferably, a patientreceiving cART or HAART prior to being treated according to the methodsof the invention is able to discontinue or reduce cART or HAARTfollowing treatment according to the methods of the invention.

For efficacy purposes, the frequency of transduced, HIV-specific CD4 Tcells, which is a novel surrogate marker for gene therapy effects, maybe determined, as discussed in more detail herein.

Compositions

In various aspects, the disclosure provides lentiviral vectors capableof delivering genetic constructs to inhibit HIV penetration ofsusceptible cells. For instance, one mechanism of action in accordanceherein is to reduce mRNA levels for CCR5 and/or CXCR4 chemokinereceptors for reducing the rates for viral entry into susceptible cells.

Alternatively, the disclosed lentiviral vectors are capable ofinhibiting the formation of HIV-infected cells by reducing the stabilityof incoming HIV genomic RNA. And in yet another embodiment, thedisclosed lentivirus vectors are capable of preventing HIV productionfrom a latently infected cell, wherein the mechanism of action is tocause instability of viral RNA sequences through the action ofinhibitory RNA including short-homology, small-interfering or otherregulatory RNA species.

The therapeutic lentiviruses disclosed generally comprise at least oneof two types of genetic cargo. First, the lentiviruses may encodegenetic elements that direct expression of small RNA capable ofinhibiting the production of chemokine receptors CCR5 and/or CXCR4 thatare important for HIV penetration of susceptible cells. The second typeof genetic cargo includes constructs capable of expressing small RNAmolecules targeting HIV RNA sequences for the purpose of preventingreverse transcription, RNA splicing, RNA translation to produceproteins, or packaging of viral genomic RNA for particle production andspreading infection. An exemplary structure is diagrammed in FIG. 3.

As shown in FIG. 3 (top panel), an exemplary construct may comprisenumerous sections or components. For example, in one embodiment, anexemplary LV construct may comprise the following sections orcomponents:

-   -   RSV—a Rous Sarcoma virus long terminal repeat;    -   5′LTR—a portion of an HIV long terminal repeat that can be        truncated to prevent replication of the vector after chromosomal        integration;    -   Psi—a packaging signal that allows for incorporation of the        vector RNA genome into viral particles during packaging;    -   RRE—a Rev Responsive element can be added to improve expression        from the transgene by mobilizing RNA out of the nucleus and into        the cytoplasm of cells;    -   cPPT—a Poly purine tract that facilitates second strand DNA        synthesis prior to integration of the transgene into the host        cell chromosome;    -   Promoter—a promoter initiates RNA transcription from the        integrated transgene to express micro-RNA clusters (or other        genetic elements of the construct), and in some embodiments, the        vectors may use an EF-1 promoter;    -   Anti-CCR5—a micro RNA targeting messenger RNA for the host cell        factor CCR5 to reduce its expression on the cell surface;    -   Anti-Rev/Tat—a micro RNA targeting HIV genomic or messenger RNA        at the junction between HIV Rev and Tat coding regions, which is        sometimes designated miRNA Tat or given a similar description in        this application;    -   Anti-Vif—a micro RNA targeting HIV genomic or messenger RNA        within the Vif coding region;    -   WPRE—a woodchuck hepatitis virus post-transcriptional regulatory        element is an additional vector component that can be used to        facilitate RNA transport of the nucleus; and    -   deltaU3 3′LTR—a modified version of a HIV 3′ long terminal        repeat where a portion of the U3 region has been deleted to        improve safety of the vector.

One of ordinary skill in the art will recognize that the abovecomponents are merely examples, and that such components may bereorganized, substituted with other elements, or otherwise changed, solong as the construct is able to prevent expression of HIV genes anddecrease the spread of infection.

Vectors of the invention may include either or both of the types ofgenetic cargo discussed above (i.e., genetic elements that directexpression of a gene or small RNAs, such as siRNA, shRNA, or miRNA thatcan prevent translation or transcription), and the vectors of theinvention may also encode additionally useful products for the purposeof treatment or diagnosis of HIV. For instance, in some embodiments,these vectors may also encode green fluorescent protein (GFP) for thepurpose of tracking the vectors or antibiotic resistance genes for thepurposes of selectively maintaining genetically-modified cells in vivo.

The combination of genetic elements incorporated into the disclosedvectors is not particularly limited. For example, a vector herein mayencode a single small RNA, two small RNAs, three small RNA, four smallRNAs, five small RNAs, six small RNAs, seven small RNAs, eight smallRNAs, nine small RNAs, or ten small RNAs, or eleven small RNAs, ortwelve small RNAs. Such vectors may additionally encode other geneticelements to function in concert with the small RNAs to preventexpression and infection of HIV.

Those of ordinary skill in the art will understand that the therapeuticlentivirus may substitute alternate sequences for the promoter region,targeting of regulatory RNA, and types of regulatory RNA. Further, thetherapeutic lentivirus of the disclosure may comprise changes in theplasmids used for packaging the lentivirus particles; these changes arerequired to increase levels of production in vitro.

Lentiviral Vector System

A lentiviral virion (particle) in accordance with various aspects andembodiments herein is expressed by a vector system encoding thenecessary viral proteins to produce a virion (viral particle). Invarious embodiments, one vector containing a nucleic acid sequenceencoding the lentiviral pol proteins is provided for reversetranscription and integration, operably linked to a promoter. In anotherembodiment, the pol proteins are expressed by multiple vectors. In otherembodiments, vectors containing a nucleic acid sequence encoding thelentiviral Gag proteins for forming a viral capsid, operably linked to apromoter, are provided. In embodiments, this gag nucleic acid sequenceis on a separate vector than at least some of the pol nucleic acidsequence. In other embodiments, the gag nucleic acid is on a separatevector from all the pol nucleic acid sequences that encode pol proteins.

Numerous modifications can be made to the vectors herein, which are usedto create the particles to further minimize the chance of obtaining wildtype revertants. These include, but are not limited to deletions of theU3 region of the LTR, tat deletions and matrix (MA) deletions. Inembodiments, the gag, pol and env vector(s) do not contain nucleotidesfrom the lentiviral genome that package lentiviral RNA, referred to asthe lentiviral packaging sequence.

The vector(s) forming the particle preferably do not contain a nucleicacid sequence from the lentiviral genome that expresses an envelopeprotein. Preferably, a separate vector that contains a nucleic acidsequence encoding an envelope protein operably linked to a promoter isused. This env vector also does not contain a lentiviral packagingsequence. In one embodiment the env nucleic acid sequence encodes alentiviral envelope protein.

In another embodiment the envelope protein is not from the lentivirus,but from a different virus. The resultant particle is referred to as apseudotyped particle. By appropriate selection of envelopes one can“infect” virtually any cell. For example, one can use an env gene thatencodes an envelope protein that targets an endocytic compartment suchas that of the influenza virus, VSV-G, alpha viruses (Semliki forestvirus, Sindbis virus), arenaviruses (lymphocytic choriomeningitisvirus), flaviviruses (tick-borne encephalitis virus, Dengue virus,hepatitis C virus, GB virus), rhabdoviruses (vesicular stomatitis virus,rabies virus), paramyxoviruses (mumps or measles) and orthomyxoviruses(influenza virus). Other envelopes that can preferably be used includethose from Moloney Leukemia Virus such as MLV-E, MLV-A and GALV. Theselatter envelopes are particularly preferred where the host cell is aprimary cell. Other envelope proteins can be selected depending upon thedesired host cell. For example, targeting specific receptors such as adopamine receptor can be used for brain delivery. Another target can bevascular endothelium. These cells can be targeted using a Filovirusenvelope. For example, the GP of Ebola, which by post-transcriptionalmodification become the GP, and GP₂ glycoproteins. In anotherembodiment, one can use different lentiviral capsids with a pseudotypedenvelope (for example, FIV or SHIV [U.S. Pat. No. 5,654,195]). A SHIVpseudotyped vector can readily be used in animal models such as monkeys.

Lentiviral vector systems as provided herein typically include at leastone helper plasmid comprising at least one of a gag, pol, or rev gene.Each of the gag, pol and rev genes may be provided on individualplasmids, or one or more genes may be provided together on the sameplasmid. In one embodiment, the gag, pol, and rev genes are provided onthe same plasmid (e.g., FIG. 4). In another embodiment, the gag and polgenes are provided on a first plasmid and the rev gene is provided on asecond plasmid (e.g., FIG. 5). Accordingly, both 3-vector (e.g., FIGS. 4and 6) and 4-vector (e.g., FIG. 5) systems can be used to produce alentivirus as described herein. In embodiments, the therapeutic vector,at least one envelope plasmid and at least one helper plasmid aretransfected into a packaging cell, for example a packaging cell line. Anon-limiting example of a packaging cell line is the 293T/17 HEK cellline. When the therapeutic vector, the envelope plasmid, and at leastone helper plasmid are transfected into the packaging cell line, alentiviral particle is ultimately produced.

In another aspect, a lentiviral vector system for expressing alentiviral particle is disclosed. The system includes a lentiviralvector as described herein; an envelope plasmid for expressing anenvelope protein optimized for infecting a cell; and at least one helperplasmid for expressing gag, pol, and rev genes, wherein when thelentiviral vector, the envelope plasmid, and the at least one helperplasmid are transfected into a packaging cell line, a lentiviralparticle is produced by the packaging cell line, wherein the lentiviralparticle is capable of inhibiting production of chemokine receptor CCR5or targeting an HIV RNA sequence.

In another aspect, the lentiviral vector, which is also referred toherein as a therapeutic vector, includes the following elements: hybrid5′ long terminal repeat (RSV/5′ LTR) (SEQ ID NOS: 34-35), Psi sequence(RNA packaging site) (SEQ ID NO: 36), RRE (Rev-response element) (SEQ IDNO: 37), cPPT (polypurine tract) (SEQ ID NO: 38), EF-1α promoter (SEQ IDNO: 4), miR30CCR5 (SEQ ID NO: 1), miR21Vif (SEQ ID NO: 2), miR185Tat(SEQ ID NO: 3), Woodchuck Post-Transcriptional Regulatory Element (WPRE)(SEQ ID NOS: 32 or 80), and ΔU3 3′ LTR (SEQ ID NO: 39). In anotheraspect, sequence variation, by way of substitution, deletion, addition,or mutation can be used to modify the sequences references herein.

In another aspect, a helper plasmid includes the following elements: CAGpromoter (SEQ ID NO: 41); HIV component gag (SEQ ID NO: 43); HIVcomponent pol (SEQ ID NO: 44); HIV Int (SEQ ID NO: 45); HIV RRE (SEQ IDNO: 46); and HIV Rev (SEQ ID NO: 47). In another aspect, the helperplasmid may be modified to include a first helper plasmid for expressingthe gag and pol genes, and a second and separate plasmid for expressingthe rev gene. In another aspect, sequence variation, by way ofsubstitution, deletion, addition, or mutation can be used to modify thesequences references herein.

In another aspect, an envelope plasmid includes the following elements:RNA polymerase II promoter (CMV) (SEQ ID NO: 60) and vesicularstomatitis virus G glycoprotein (VSV-G) (SEQ ID NO: 62). In anotheraspect, sequence variation, by way of substitution, deletion, addition,or mutation can be used to modify the sequences references herein.

In various aspects, the plasmids used for lentiviral packaging aremodified by substitution, addition, subtraction or mutation of variouselements without loss of vector function. For example, and withoutlimitation, the following elements can replace similar elements in theplasmids that comprise the packaging system: Elongation Factor-1 (EF-1),phosphoglycerate kinase (PGK), and ubiquitin C (UbC) promoters canreplace the CMV or CAG promoter. SV40 poly A and bGH poly A can replacethe rabbit beta globin poly A. The HIV sequences in the helper plasmidcan be constructed from different HIV strains or clades. The VSV-Gglycoprotein can be substituted with membrane glycoproteins from felineendogenous virus (RD114), gibbon ape leukemia virus (GALV), Rabies(FUG), lymphocytic choriomeningitis virus (LCMV), influenza A fowlplague virus (FPV), Ross River alphavirus (RRV), murine leukemia virus10A1 (MLV), or Ebola virus (EboV).

Various lentiviral packaging systems can be acquired commercially (e.g.,Lenti-vpak packaging kit from OriGene Technologies, Inc., Rockville,Md.), and can also be designed as described herein. Moreover, it iswithin the skill of a person ordinarily skilled in the art to substituteor modify aspects of a lentiviral packaging system to improve any numberof relevant factors, including the production efficiency of a lentiviralparticle.

Bioassays

In various aspects, the present invention includes bioassays fordetermining the success of HIV treatment for achieving a functionalcure. These assays provide a method for measuring the efficacy of thedisclosed methods of immunization and treatment by measuring thefrequency of transduced, HIV specific CD4 T cells in a patient.HIV-specific CD4 T cells are recognizable because, among others, theyproliferate, change the composition of cell surface markers, inducesignaling pathways including phosphorylation, and/or express specificmarker proteins that may be cytokines, chemokines, caspases,phosphorylated signaling molecules or other cytoplasmic and/or nuclearcomponents. Specific responding CD4 T cells are recognized for example,using labeled monoclonal antibodies or specific in situ amplification ofmRNA sequences, that allow sorting of HIV-specific cells using flowcytometry sorting, magnetic bead separation or other recognized methodsfor antigen-specific CD4 T cell isolation. The isolated CD4 T cells aretested to determine the frequency of cells carrying integratedtherapeutic lentivirus. Single cell testing methods may also be usedincluding microfluidic separation of individual cells that are coupledwith mass spectrometry, PCR, ELISA or antibody staining to confirmresponsiveness to HIV and presence of integrated therapeutic lentivirus.

Thus, in various embodiments, following application of a treatmentaccording to the invention (e.g., (a) immunization, (b) ex vivoleukocyte/lymphocyte culture; (c) re-stimulation with purified proteins,inactivated viruses, virally vectored proteins, bacterially vectoredproteins, biological or chemical adjuvants including cytokines and/orchemokines, vehicles; and (d) infusion of the enriched, transduced Tcells), a patient may be subsequently assayed to determine the efficacyof the treatment. A threshold value of target T cells in the body may beestablished to measure a functional cure at a determined value, forexample, at about 1×10⁸ HIV-specific CD4 T cells bearing geneticmodification from therapeutic lentivirus. Alternatively, the thresholdvalue may be about 1×10⁵, about 1×10⁶, about 1×10⁷, about 1×10⁸, about1×10⁹, or about 1×10¹⁰ CD4 T cells in the body of the patient.

HIV-specific CD4 T cells bearing genetic modification from therapeuticlentivirus can be determined using any suitable method, such as but notlimited to flow cytometry, cell sorting, FACS analysis, DNA cloning,PCR, RT-PCR or Q-PCR, ELISA, FISH, western blotting, southern blotting,high throughput sequencing, RNA sequencing, oligonucleotide primerextension, or other methods known in the art.

While methods for defining antigen specific T cells with geneticmodifications are known in the art, utilizing such methods to combineidentifying HIV-specific T cells with integrated or non-integrated genetherapy constructs as a standard measure for efficacy is a novel conceptin the field of HIV treatment, as described variously herein.

Doses and Dosage Forms

The disclosed methods and compositions can be used for treating HIV+patients during various stages of their disease. Accordingly, dosingregimens may vary based upon the condition of the patient and the methodof administration.

In various embodiments, HIV-specific vaccines for the initial in vivoimmunization are administered to a subject in need in varying doses. Ingeneral, vaccines delivered by intramuscular injection include about 10μg to about 300 μg, about 25 μg to about 275 μg, about 50 μg to about250 μg, about 75 μg to about 225, or about 100 μg to about 200 μg of HIVprotein, either total virus protein prepared from inactivated virusparticles, virus-like particles or purified virus protein fromrecombinant systems or purified from virus preparations. Recombinantviral or bacterial vectors may be administered by any and all of theroutes described. Intramuscular vaccines will include about 1 μg toabout 100 μg, about 10 μg to about 90 μg, about 20 μg to about 80 μg,about 30 μg to about 70 μg, about 40 μg to about 60 μg, or about 50 μgof suitable adjuvant molecules and be suspended in oil, saline, bufferor water in volumes of 0.1 to 5 ml per injection dose, and may besoluble or emulsion preparations. Vaccines delivered orally, rectally,bucally, at genital mucosal or intranasally, including somevirally-vectored or bacterially-vectored vaccines, fusion proteins,liposome formulations or similar preparations, may contain higheramounts of virus protein and adjuvant. Dermal, sub-dermal orsubcutaneous vaccines utilize protein and adjuvant amounts more similarto oral, rectal or intranasal-delivered vaccines. Depending on responsesto the initial immunization, vaccination may be repeated 1-5 times usingthe same or alternate routes for delivery. Intervals may be of 2-24weeks between immunizations. Immune responses to vaccination aremeasured by testing HIV-specific antibodies in serum, plasma, vaginalsecretions, rectal secretions, saliva or bronchoalveolar lavage fluids,using ELISA or similar methodology. Cellular immune responses are testedby in vitro stimulation with vaccine antigens followed by staining forintracellular cytokine accumulation followed by flow cytometry orsimilar methods including lymphoproliferation, expression ofphosphorylated signaling proteins or changes in cell surface activationmarkers. Upper limits of dosing may be determined based on theindividual patient and will depend on toxicity/safety profiles for eachindividual product or product lot.

Immunization may occur once, twice, three times, or repeatedly. Forinstance, an agent for HIV immunization may be administered to a subjectin need once a week, once every other week, once every three weeks, oncea month, every other month, every three months, every six months, everynine months, once a year, every eighteen months, every two years, every36 months, or every three years.

Immunization will generally occur at least once before ex vivo expansionand enrichment of CD4 T cells, and immunization may occur once, twice,three times, or more after ex vivo leukocyte/lymphocyteculture/re-stimulation and infusion.

In one embodiment, HIV vaccines for immunization are administered as apharmaceutical composition. In one embodiment, the pharmaceuticalcomposition comprising an HIV vaccine is formulated in a wide variety ofnasal, pulmonary, oral, topical, or parenteral dosage forms for clinicalapplication. Each of the dosage forms can comprise variousdisintegrating agents, surfactants, fillers, thickeners, binders,diluents such as wetting agents or other pharmaceutically acceptableexcipients. The pharmaceutical composition comprising an HIV vaccine canalso be formulated for injection.

HIV vaccine compositions for the purpose of immunization can beadministered using any pharmaceutically acceptable method, such asintranasal, buccal, sublingual, oral, rectal, ocular, parenteral(intravenously, intradermally, intramuscularly, subcutaneously,intracistemally, intraperitoneally), pulmonary, intravaginal, locallyadministered, topically administered, topically administered afterscarification, mucosally administered, via an aerosol, or via a buccalor nasal spray formulation.

Further, the HIV vaccine compositions can be formulated into anypharmaceutically acceptable dosage form, such as a solid dosage form,tablet, pill, lozenge, capsule, liquid dispersion, gel, aerosol,pulmonary aerosol, nasal aerosol, ointment, cream, semi-solid dosageform, and a suspension. Further, the composition may be a controlledrelease formulation, sustained release formulation, immediate releaseformulation, or any combination thereof. Further, the composition may bea transdermal delivery system.

In another embodiment, the pharmaceutical composition comprising an HIVvaccine is formulated in a solid dosage form for oral administration,and the solid dosage form can be powders, granules, capsules, tablets orpills. In yet another embodiment, the solid dosage form includes one ormore excipients such as calcium carbonate, starch, sucrose, lactose,microcrystalline cellulose or gelatin. In addition, the solid dosageform can include, in addition to the excipients, a lubricant such astalc or magnesium stearate. In some embodiments, the oral dosage form isin immediate release or a modified release form. Modified release dosageforms include controlled or extended release, enteric release, and thelike. The excipients used in the modified release dosage forms arecommonly known to a person of ordinary skill in the art.

In a further embodiment, the pharmaceutical composition comprising a HIVvaccine is formulated as a sublingual or buccal dosage form. Such dosageforms comprise sublingual tablets or solution compositions that areadministered under the tongue and buccal tablets that are placed betweenthe cheek and gum.

In yet a further embodiment, the pharmaceutical composition comprisingan HIV vaccine is formulated as a nasal dosage form. Such dosage formsof the present invention comprise solution, suspension, and gelcompositions for nasal delivery.

In one embodiment, the pharmaceutical composition is formulated in aliquid dosage form for oral administration, such as suspensions,emulsions or syrups. In other embodiments, the liquid dosage form caninclude, in addition to commonly used simple diluents such as water andliquid paraffin, various excipients such as humectants, sweeteners,aromatics or preservatives. In particular embodiments, the compositioncomprising HIV vaccine or a pharmaceutically acceptable salt thereof isformulated to be suitable for administration to a pediatric patient.

In one embodiment, the pharmaceutical composition is formulated in adosage form for parenteral administration, such as sterile aqueoussolutions, suspensions, emulsions, non-aqueous solutions orsuppositories. In other embodiments, the non-aqueous solutions orsuspensions includes propyleneglycol, polyethyleneglycol, vegetable oilssuch as olive oil or injectable esters such as ethyl oleate. As a basefor suppositories, witepsol, macrogol, tween 61, cacao oil, laurin oilor glycerinated gelatin can be used.

The dosage of the pharmaceutical composition can vary depending on thepatient's weight, age, gender, administration time and mode, excretionrate, and the severity of disease.

For the purposes of re-stimulation, lymphocytes, PBMCs, and/or CD4 Tcells are generally removed from a patient and isolated forre-stimulation and culturing. The isolated cells may be contacted withthe same HIV vaccine or activating agent used for immunization or adifferent HIV vaccine or activating agent. In one embodiment, theisolated cells are contacted with about 10 ng to 5 μg of an HIV vaccineor activating agent per about 10⁶ cells in culture (or any othersuitable amount). More specifically, the isolated cells may be contactedwith about 50 ng, about 100 ng, about 200 ng, about 300 ng, about 400ng, about 500 ng, about 600 ng, about 700 ng, about 800 ng, about 900ng, about 1 μg, about 1.5 μg, about 2 μg, about 2.5 μg, about 3 μg,about 3.5 μg, about 4 μg, about 4.5 μg, or about 5 μg of an HIV vaccineor activating agent per about 10⁶ cells in culture.

Activating agents or vaccines are generally used once for each in vitrocell culture but may be repeated after intervals of about 15 to about 35days. For example, a repeat dosing could occur at about 15, about 16,about 17, about 18, about 19, about 20, about 21, about 22, about 23,about 24, about 25, about 26, about 27, about 28, about 29, about 30,about 31, about 32, about 33, about 34, or about 35 days.

For transduction of the enriched, re-stimulated cells, the cells may betransduced with lentiviral vectors or with other known vector systems asdisclosed, for example, in FIG. 4 or

FIG. 6. The cells being transduced may be contacted with about 1-1,000viral genomes (measured by RT-PCR assay of culture fluids containinglentivirus vector) per target cell in culture (or any other suitableamount). Lentivirus transduction may be repeated 1-5 times using thesame range of 1-1,000 viral genomes per target cell in culture.

Cellular Enrichment

In various embodiments, cells such as T cells are obtained from an HIVinfected patient and cultured. Culturing can occur in multiwell platesin a culture medium comprising conditioned media (“CM”). The levels ofsupernatant p24^(gag) (“p24”) and viral RNA levels may be assessed bystandard means. Those patients whose CM-cultured cells have peak p24supernatant levels of less than 1 ng/ml may be suitable patients forlarge-scale T-cell expansion in CM with or without the use of additionalanti-viral agents. Additionally, different drugs or drug combinations ofinterest may be added to different wells and the impact on virus levelsin the sample may be assessed by standard means. Those drug combinationsproviding adequate viral suppression are therapeutically usefulcombinations. It is within the capacity of a competent technician todetermine what constitutes adequate viral suppression in relation to aparticular subject. In order to test the effectiveness of drugs ofinterest in limiting viral expansion, additional factors such asanti-CD3 antibodies may be added to the culture to stimulate viralproduction. Unlike culture methods for HIV infected cell samples knownin the art, CM allows the culture of T cells for periods of over twomonths, thereby providing an effective system in which to assay longterm drug effectiveness.

This approach allows the inhibition of gene expression driven by the HIVLTR promoter region in a cell population by the culture of cells in amedium comprising the CM. Culture in CM4 likely inhibits HIV LTR drivengene expression by altering one or more interactions betweentranscription mediating proteins and HIV gene expression regulatoryelements. Transcription-mediating proteins of interest include host cellencoded proteins such as AP-1, NFkappaB, NF-AT, IRF, LEF-1 and Spl, andthe HIV encoded protein Tat. HIV gene expression regulatory elements ofinterest include binding sites for AP-1, NFkappaB, NF-AT, IRF, LEF-1 andSpl, as well as the transacting responsive element (“TAR”) whichinteracts with Tat.

In a preferred embodiment, the HIV infected cells are obtained from asubject with susceptible transcription mediating protein sequences andsusceptible HIV regulatory element sequences. In a more preferredembodiment, the HIV infected cells are obtained from a subject havingwild-type transcription-mediating protein sequences and wild-type HIVregulatory sequences.

Another method of enriching T Cells utilizes immunoaffinity-basedselection. This method includes the simultaneous enrichment or selectionof a first and second population of cells, such as a CD4+ and CD8+ cellpopulation. Cells containing primary human T cells are contacted with afirst immunoaffinity reagent that specifically binds to CD4 and a secondimmunoaffinity reagent that specifically binds to CD8 in an incubationcomposition, under conditions whereby the immunoaffinity reagentsspecifically bind to CD4 and CD8 molecules, respectively, on the surfaceof cells in the sample. Cells bound to the first and/or the secondimmunoaffinity reagent are recovered, thereby generating an enrichedcomposition comprising CD4+ cells and CD8+ cells. This approach mayinclude incubation of the composition with a concentration of the firstand/or second immunoaffinity reagent that is at a sub-optimal yieldconcentration. Notably, in some embodiments, transduced cells are amixed T cell population, and in other embodiments transduced cells arenot a mixed T cell population.

In some embodiments, immunoaffinity-based selection is used where thesolid support is a sphere, such as a bead, such as a microbead ornanobead. In other embodiments, the bead can be a magnetic bead. Inanother embodiment, the antibody contains one or more binding partnerscapable of forming a reversible bond with a binding reagent immobilizedon the solid surface, such as a sphere or chromatography matrix, whereinthe antibody is reversibly mobilized to the solid surface. In someembodiments, cells expressing a cell surface marker bound by theantibody on said solid surface are capable of being recovered from thematrix by disruption of the reversible binding between the bindingreagent and binding partner. In some embodiments, the binding reagent isstreptavidin or is a streptavidin analog or mutant. In some embodiments,immunoaffinity-based selection is used to capture cells specificallyresponding to HIV proteins, vaccines or peptides based on expression ofcytokines in these cells. A bi-specific capture reagent binds to T cellsand also captures cytokine released from that cell. The cytokine ispreferably interferon gamma but may include tumor necrosis factor alphaor other cytokines known to be produced by T cells. The immobilizedcytokine is recognized by a second immune-affinity reagent that ismodified by a magnetic bead. Cells with the first capturereagent-cytokine-second immune-affinity reagent and magnetic bead areretained on a magnetic column and are thus purified away from cells thatdid not express cytokine after HIV protein, vaccine or peptidestimulation and are maintained in a viable state. Removing the matrixfrom a magnetic field allows release of the labeled cells and capture asa highly enriched population. In some cases, the enriched cells may becultured for 1-30 days to increase in number before being stimulatedwith a polyclonal mitogen such as anti-CD³/anti-CD28 microbeads orsimilar stimulation reagents that are compatible with lentivirustransduction.

Stable transduction of primary cells of the hematopoietic system and/orhematopoietic stem cells may be obtained by contacting, in vitro or exvivo, the surface of the cells with both a lentiviral vector and atleast one molecule which binds the cell surface. The cells may becultured in a ventilated vessel comprising two or more layers underconditions conducive to growth and/or proliferation. In someembodiments, this approach may be used in conjunction with non-CD4+ Tcell depletion and/or broad polyclonal expansion.

In another approach to T cell enrichment, PBMCs are stimulated with apeptide and enriched for cells secreting a cytokine, such asinterferon-gamma. This approach generally involves stimulating a mixtureof cells containing T cells with antigen, and effecting a separation ofantigen-stimulated cells according to the degree to which they arelabeled with the product. Antigen stimulation is achieved by exposingthe cells to at least one antigen under conditions effective to elicitantigen-specific stimulation of at least one T cell. Labeling with theproduct is achieved by modifying the surface of the cells to contain atleast one capture moiety, culturing the cells under conditions in whichthe product is secreted, released and specifically bound (“captured” or“entrapped”) to said capture moiety; and labeling the captured productwith a label moiety, where the labeled cells are not lysed as part ofthe labeling procedure or as part of the separation procedure. Thecapture moiety may incorporate detection of cell surface glycoproteinsCD3 or CD4 to refine the enrichment step and increase the proportion ofantigen-specific T cells in general, of CD4+ T cells in specific.

The following examples are given to illustrate aspects of the presentinvention. It should be understood, however, that the invention is notto be limited to the specific conditions or details described in theseexamples. All printed publications referenced herein are specificallyincorporated by reference.

EXAMPLES Example 1: Development of a Lentiviral Vector System

A lentiviral vector system was developed as summarized in FIG. 3 (linearform) and FIG. 4 (circularized form). Referring first to the top portionof FIG. 3, a representative therapeutic vector has been designed andproduced with the following elements being from left to right: hybrid 5′long terminal repeat (RSV/5′ LTR) (SEQ ID NOS: 34-35), Psi sequence (RNApackaging site) (SEQ ID NO: 36), RRE (Rev-response element) (SEQ ID NO:37), cPPT (polypurine tract) (SEQ ID NO: 38), EF-1α promoter (SEQ ID NO:4), miR30CCR5 (SEQ ID NO: 1), miR21Vif (SEQ ID NO: 2), miR185Tat (SEQ IDNO: 3), Woodchuck Post-Transcriptional Regulatory Element (WPRE) (SEQ IDNOS: 32 or 80), and ΔU3 3′ LTR (SEQ ID NO: 39). The therapeutic vectordetailed in FIG. 3 is also referred to herein as AGT103.

Referring next to the middle portion of FIG. 3, a helper plasmid hasbeen designed and produced with the following elements being from leftto right: CAG promoter (SEQ ID NO: 41); HIV component gag (SEQ ID NO:43); HIV component pol (SEQ ID NO: 44); HIV Int (SEQ ID NO: 45); HIV RRE(SEQ ID NO: 46); and HIV Rev (SEQ ID NO: 47).

Referring next to the lower portion of FIG. 3, an envelope plasmid hasbeen designed and produced with the following elements being from leftto right: RNA polymerase II promoter (CMV) (SEQ ID NO: 60) and vesicularstomatitis virus G glycoprotein (VSV-G) (SEQ ID NO: 62).

Lentiviral particles were produced in 293T/17 HEK cells (purchased fromAmerican Type Culture Collection, Manassas, Va.) following transfectionwith the therapeutic vector, the envelope plasmid, and the helperplasmid (as shown in FIG. 3). The transfection of 293T/17 HEK cells,which produced functional viral particles, employed the reagentPoly(ethylenimine) (PEI) to increase the efficiency of plasmid DNAuptake. The plasmids and DNA were initially added separately in culturemedium without serum in a ratio of 3:1 (mass ratio of PEI to DNA). After2-3 days, cell medium was collected and lentiviral particles werepurified by high-speed centrifugation and/or filtration followed byanion-exchange chromatography. The concentration of lentiviral particlescan be expressed in terms of transducing units/ml (TU/ml). Thedetermination of TU was accomplished by measuring HIV p24 levels inculture fluids (p24 protein is incorporated into lentiviral particles),measuring the number of viral DNA copies per cell by quantitative PCR,or by infecting cells and using light (if the vectors encode luciferaseor fluorescent protein markers).

As mentioned above, a 3-vector system (i.e., a 2-vector lentiviralpackaging system) was designed for the production of lentiviralparticles. A schematic of the 3-vector system is shown in FIG. 4. Theschematic of FIG. 4 is a circularized version of the linear systempreviously described in FIG. 3. Briefly, and with reference to FIG. 4,the top-most vector is a helper plasmid, which, in this case, includesRev. The vector appearing in the middle of FIG. 4 is the envelopeplasmid. The bottom-most vector is the previously described therapeuticvector.

Referring more specifically to FIG. 4, the Helper plus Rev plasmidincludes a CAG enhancer (SEQ ID NO: 40); a CAG promoter (SEQ ID NO: 41);a chicken beta actin intron (SEQ ID NO: 42); a HIV gag (SEQ ID NO: 43);a HIV Pol (SEQ ID NO: 44); a HIV Int (SEQ ID NO: 45); a HIV RRE (SEQ IDNO: 46); a HIV Rev (SEQ ID NO: 47); and a rabbit beta globin poly A (SEQID NO: 48).

The Envelope plasmid includes a CMV promoter (SEQ ID NO: 60); a betaglobin intron (SEQ ID NO: 61); a VSV-G (SEQ ID NO: 62); and a rabbitbeta globin poly A (SEQ ID NO: 63).

In an alternate vector system, and with respect to FIG. 6, the vectorsequences are provided herein as SEQ ID NOs: 105-107.

Synthesis of a 2-Vector Lentiviral Packaging System Including Helper(Plus Rev) and Envelope plasmids.

Materials and Methods:

Construction of the Helper Plasmid:

The helper plasmid was constructed by initial PCR amplification of a DNAfragment from the pNL4-3 HIV plasmid (NIH Aids Reagent Program)containing Gag, Pol, and Integrase genes. Primers were designed toamplify the fragment with EcoRI and NotI restriction sites which couldbe used to insert at the same sites in the pCDNA3 plasmid (Invitrogen).The forward primer was (5′-TAAGCAGAATTC ATGAATTTGCCAGGAAGAT-3′) (SEQ IDNO: 81) and reverse primer was(5′-CCATACAATGAATGGACACTAGGCGGCCGCACGAAT-3′) (SEQ ID NO: 82). Thesequence for the Gag, Pol, Integrase fragment was as follows:

(SEQ ID NO: 83) GAATTCATGAATTTGCCAGGAAGATGGAAACCAAAAATGATAGGGGGAATTGGAGGTTTTATCAAAGTAAGACAGTATGATCAGATACTCATAGAAATCTGCGGACATAAAGCTATAGGTACAGTATTAGTAGGACCTACACCTGTCAACATAATTGGAAGAAATCTGTTGACTCAGATTGGCTGCACTTTAAATTTTCCCATTAGTCCTATTGAGACTGTACCAGTAAAATTAAAGCCAGGAATGGATGGCCCAAAAGTTAAACAATGGCCATTGACAGAAGAAAAAATAAAAGCATTAGTAGAAATTTGTACAGAAATGGAAAAGGAAGGAAAAATTTCAAAAATTGGGCCTGAAAATCCATACAATACTCCAGTATTTGCCATAAAGAAAAAAGACAGTACTAAATGGAGAAAATTAGTAGATTTCAGAGAACTTAATAAGAGAACTCAAGATTTCTGGGAAGTTCAATTAGGAATACCACATCCTGCAGGGTTAAAACAGAAAAAATCAGTAACAGTACTGGATGTGGGCGATGCATATTTTTCAGTTCCCTTAGATAAAGACTTCAGGAAGTATACTGCATTTACCATACCTAGTATAAACAATGAGACACCAGGGATTAGATATCAGTACAATGTGCTTCCACAGGGATGGAAAGGATCACCAGCAATATTCCAGTGTAGCATGACAAAAATCTTAGAGCCTTTTAGAAAACAAAATCCAGACATAGTCATCTATCAATACATGGATGATTTGTATGTAGGATCTGACTTAGAAATAGGGCAGCATAGAACAAAAATAGAGGAACTGAGACAACATCTGTTGAGGTGGGGATTTACCACACCAGACAAAAAACATCAGAAAGAACCTCCATTCCTTTGGATGGGTTATGAACTCCATCCTGATAAATGGACAGTACAGCCTATAGTGCTGCCAGAAAAGGACAGCTGGACTGTCAATGACATACAGAAATTAGTGGGAAAATTGAATTGGGCAAGTCAGATTTATGCAGGGATTAAAGTAAGGCAATTATGTAAACTTCTTAGGGGAACCAAAGCACTAACAGAAGTAGTACCACTAACAGAAGAAGCAGAGCTAGAACTGGCAGAAAACAGGGAGATTCTAAAAGAACCGGTACATGGAGTGTATTATGACCCATCAAAAGACTTAATAGCAGAAATACAGAAGCAGGGGCAAGGCCAATGGACATATCAAATTTATCAAGAGCCATTTAAAAATCTGAAAACAGGAAAGTATGCAAGAATGAAGGGTGCCCACACTAATGATGTGAAACAATTAACAGAGGCAGTACAAAAAATAGCCACAGAAAGCATAGTAATATGGGGAAAGACTCCTAAATTTAAATTACCCATACAAAAGGAAACATGGGAAGCATGGTGGACAGAGTATTGGCAAGCCACCTGGATTCCTGAGTGGGAGTTTGTCAATACCCCTCCCTTAGTGAAGTTATGGTACCAGTTAGAGAAAGAACCCATAATAGGAGCAGAAACTTTCTATGTAGATGGGGCAGCCAATAGGGAAACTAAATTAGGAAAAGCAGGATATGTAACTGACAGAGGAAGACAAAAAGTTGTCCCCCTAACGGACACAACAAATCAGAAGACTGAGTTACAAGCAATTCATCTAGCTTTGCAGGATTCGGGATTAGAAGTAAACATAGTGACAGACTCACAATATGCATTGGGAATCATTCAAGCACAACCAGATAAGAGTGAATCAGAGTTAGTCAGTCAAATAATAGAGCAGTTAATAAAAAAGGAAAAAGTCTACCTGGCATGGGTACCAGCACACAAAGGAATTGGAGGAAATGAACAAGTAGATAAATTGGTCAGTGCTGGAATCAGGAAAGTACTATTTTTAGATGGAATAGATAAGGCCCAAGAAGAACATGAGAAATATCACAGTAATTGGAGAGCAATGGCTAGTGATTTTAACCTACCACCTGTAGTAGCAAAAGAAATAGTAGCCAGCTGTGATAAATGTCAGCTAAAAGGGGAAGCCATGCATGGACAAGTAGACTGTAGCCCAGGAATATGGCAGCTAGATTGTACACATTTAGAAGGAAAAGTTATCTTGGTAGCAGTTCATGTAGCCAGTGGATATATAGAAGCAGAAGTAATTCCAGCAGAGACAGGGCAAGAAACAGCATACTTCCTCTTAAAATTAGCAGGAAGATGGCCAGTAAAAACAGTACATACAGACAATGGCAGCAATTTCACCAGTACTACAGTTAAGGCCGCCTGTTGGTGGGCGGGGATCAAGCAGGAATTTGGCATTCCCTACAATCCCCAAAGTCAAGGAGTAATAGAATCTATGAATAAAGAATTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTTAAGACAGCAGTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTTGGAAAGGACCAGCAAAGCTCCTCTGGAAAGGTGAAGGGGCAGTAGTAATACAAGATAATAGTGACATAAAAGTAGTGCCAAGAAGAAAAGCAAAGATCATCAGGGATTATGGAAAACAGATGGCAGGTGATGATTGTGTGGCAAGTAGACAGGATGAGGATTAA

Next, a DNA fragment containing the Rev, RRE, and rabbit beta globinpoly A sequence with XbaI and XmaI flanking restriction sites wassynthesized by MWG Operon. The DNA fragment was then inserted into theplasmid at the XbaI and XmaI restriction sites The DNA sequence was asfollows:

(SEQ ID NO: 84) TCTAGAATGGCAGGAAGAAGCGGAGACAGCGACGAAGAGCTCATCAGAACAGTCAGACTCATCAAGCTTCTCTATCAAAGCAACCCACCTCCCAATCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCCTTGGCACTTATCTGGGACGATCTGCGGAGCCTGTGCCTCTTCAGCTACCACCGCTTGAGAGACTTACTCTTGATTGTAACGAGGATTGTGGAACTTCTGGGACGCAGGGGGTGGGAAGCCCTCAAATATTGGTGGAATCTCCTACAATATTGGAGTCAGGAGCTAAAGAATAGAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCATATGCTGGCTGCCATGAACAAAGGTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGAAGATCCCTCGACCTGCAGCCCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCGGATCCGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCAGCGGCCGCCCCGGG

Finally, the CMV promoter of pCDNA3.1 was replaced with the CAGenhancer/promoter plus a chicken beta actin intron sequence. A DNAfragment containing the CAG enhancer/promoter/intron sequence with MluIand EcoRI flanking restriction sites was synthesized by MWG Operon. TheDNA fragment was then inserted into the plasmid at the MluI and EcoRIrestriction sites. The DNA sequence was as follows:

(SEQ ID NO: 85) ACGCGTTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTCGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTAAAGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGGCGGTCGGGCTGTAACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTGCGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCATCTCCAGCCTCGGGGCTGCCGCAGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTG ACCGGCGGGAATTC

Construction of the VSV-G Envelope Plasmid:

The vesicular stomatitis Indiana virus glycoprotein (VSV-G) sequence wassynthesized by MWG Operon with flanking EcoRI restriction sites. The DNAfragment was then inserted into the pCDNA3.1 plasmid (Invitrogen) at theEcoRI restriction site and the correct orientation was determined bysequencing using a CMV specific primer. The DNA sequence was as follows:

(SEQ ID NO: 86) GAATTCATGAAGTGCCTTTTGTACTTAGCCTTTTTATTCATTGGGGTGAATTGCAAGTTCACCATAGTTTTTCCACACAACCAAAAAGGAAACTGGAAAAATGTTCCTTCTAATTACCATTATTGCCCGTCAAGCTCAGATTTAAATTGGCATAATGACTTAATAGGCACAGCCTTACAAGTCAAAATGCCCAAGAGTCACAAGGCTATTCAAGCAGACGGTTGGATGTGTCATGCTTCCAAATGGGTCACTACTTGTGATTTCCGCTGGTATGGACCGAAGTATATAACACATTCCATCCGATCCTTCACTCCATCTGTAGAACAATGCAAGGAAAGCATTGAACAAACGAAACAAGGAACTTGGCTGAATCCAGGCTTCCCTCCTCAAAGTTGTGGATATGCAACTGTGACGGATGCCGAAGCAGTGATTGTCCAGGTGACTCCTCACCATGTGCTGGTTGATGAATACACAGGAGAATGGGTTGATTCACAGTTCATCAACGGAAAATGCAGCAATTACATATGCCCCACTGTCCATAACTCTACAACCTGGCATTCTGACTATAAGGTCAAAGGGCTATGTGATTCTAACCTCATTTCCATGGACATCACCTTCTTCTCAGAGGACGGAGAGCTATCATCCCTGGGAAAGGAGGGCACAGGGTTCAGAAGTAACTACTTTGCTTATGAAACTGGAGGCAAGGCCTGCAAAATGCAATACTGCAAGCATTGGGGAGTCAGACTCCCATCAGGTGTCTGGTTCGAGATGGCTGATAAGGATCTCTTTGCTGCAGCCAGATTCCCTGAATGCCCAGAAGGGTCAAGTATCTCTGCTCCATCTCAGACCTCAGTGGATGTAAGTCTAATTCAGGACGTTGAGAGGATCTTGGATTATTCCCTCTGCCAAGAAACCTGGAGCAAAATCAGAGCGGGTCTTCCAATCTCTCCAGTGGATCTCAGCTATCTTGCTCCTAAAAACCCAGGAACCGGTCCTGCTTTCACCATAATCAATGGTACCCTAAAATACTTTGAGACCAGATACATCAGAGTCGATATTGCTGCTCCAATCCTCTCAAGAATGGTCGGAATGATCAGTGGAACTACCACAGAAAGGGAACTGTGGGATGACTGGGCACCATATGAAGACGTGGAAATTGGACCCAATGGAGTTCTGAGGACCAGTTCAGGATATAAGTTTCCTTTATACATGATTGGACATGGTATGTTGGACTCCGATCTTCATCTTAGCTCAAAGGCTCAGGTGTTCGAACATCCTCACATTCAAGACGCTGCTTCGCAACTTCCTGATGATGAGAGTTTATTTTTTGGTGATACTGGGCTATCCAAAAATCCAATCGAGCTTGTAGAAGGTTGGTTCAGTAGTTGGAAAAGCTCTATTGCCTCTTTTTTCTTTATCATAGGGTTAATCATTGGACTATTCTTGGTTCTCCGAGTTGGTATCCATCTTTGCATTAAATTAAAGCACACCAAGAAAAGACAGATTTATACAGACATAGAGATGAGAATTC

A 4-vector system (i.e., a 3-vector lentiviral packaging system) hasalso been designed and produced using the methods and materialsdescribed herein. A schematic of the 4-vector system is shown in FIG. 5.Briefly, and with reference to FIG. 5, the top-most vector is a helperplasmid, which, in this case, does not include Rev. The vector secondfrom the top is a separate Rev plasmid. The vector second from thebottom is the envelope plasmid. The bottom-most vector is the previouslydescribed therapeutic vector.

Referring, in part, to FIG. 5, the Helper plasmid includes a CAGenhancer (SEQ ID NO: 49); a CAG promoter (SEQ ID NO: 50); a chicken betaactin intron (SEQ ID NO: 51); a HIV gag (SEQ ID NO: 52); a HIV Pol (SEQID NO: 53); a HIV Int (SEQ ID NO: 54); a HIV RRE (SEQ ID NO: 55); and arabbit beta globin poly A (SEQ ID NO: 56).

The Rev plasmid includes a RSV promoter (SEQ ID NO: 57); a HIV Rev (SEQID NO: 58); and a rabbit beta globin poly A (SEQ ID NO: 59).

The Envelope plasmid includes a CMV promoter (SEQ ID NO: 60); a betaglobin intron (SEQ ID NO: 61); a VSV-G (SEQ ID NO: 62); and a rabbitbeta globin poly A (SEQ ID NO: 63).

Synthesis of a 3-Vector Lentiviral Packaging System Including Helper,Rev, and Envelope Plasmids.

Materials and Methods:

Construction of the Helper Plasmid without Rev:

The Helper plasmid without Rev was constructed by inserting a DNAfragment containing the RRE and rabbit beta globin poly A sequence. Thissequence was synthesized by MWG Operon with flanking XbaI and XmaIrestriction sites. The RRE/rabbit poly A beta globin sequence was theninserted into the Helper plasmid at the XbaI and XmaI restriction sites.The DNA sequence is as follows:

(SEQ ID NO: 87) TCTAGAAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCATATGCTGGCTGCCATGAACAAAGGTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGAAGATCCCTCGACCTGCAGCCCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCGGATCCGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCACCCGGG

Construction of the Rev Plasmid:

The RSV promoter and HIV Rev sequence was synthesized as a single DNAfragment by MWG Operon with flanking MfeI and XbaI restriction sites.The DNA fragment was then inserted into the pCDNA3.1 plasmid(Invitrogen) at the MfeI and XbaI restriction sites in which the CMVpromoter is replaced with the RSV promoter. The DNA sequence was asfollows:

(SEQ ID NO: 88) CAATTGCGATGTACGGGCCAGATATACGCGTATCTGAGGGGACTAGGGTGTGTTTAGGCGAAAAGCGGGGCTTCGGTTGTACGCGGTTAGGAGTCCCCTCAGGATATAGTAGTTTCGCTTTTGCATAGGGAGGGGGAAATGTAGTCTTATGCAATACACTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACAGGTCTGACATGGATTGGACGAACCACTGAATTCCGCATTGCAGAGATAATTGTATTTAAGTGCCTAGCTCGATACAATAAACGCCATTTGACCATTCACCACATTGGTGTGCACCTCCAAGCTCGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCCCTCGAAGCTAGCGATTAGGCATCTCCTATGGCAGGAAGAAGCGGAGACAGCGACGAAGAACTCCTCAAGGCAGTCAGACTCATCAAGTTTCTCTATCAAAGCAACCCACCTCCCAATCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCCTTAGCACTTATCTGGGACGATCTGCGGAGCCTGTGCCTCTTCAGCTACCACCGCTTGAGAGACTTACTCTTGATTGTAACGAGGATTGTGGAACTTCTGGGACGCAGGGGGTGGGAAGCCCTCAAATATTGGTGGAATCTCCTACAATATTGGAGTCAGGAGCTAAAGAATAGTCTAGA

The plasmids for the 2-vector and 3-vector packaging systems could bemodified with similar elements and the intron sequences couldpotentially be removed without loss of vector function. For example, thefollowing elements could replace similar elements in the 2-vector and3-vector packaging system:

Promoters: Elongation Factor-1 (EF-1) (SEQ ID NO: 64), phosphoglyceratekinase (PGK) (SEQ ID NO: 65), and ubiquitin C (UbC) (SEQ ID NO: 66) canreplace the CMV (SEQ ID NO: 60) or CAG promoter (SEQ ID NO: 100). Thesesequences can also be further varied by addition, substitution, deletionor mutation.

Poly A sequences: SV40 poly A (SEQ ID NO: 67) and bGH poly A (SEQ ID NO:68) can replace the rabbit beta globin poly A (SEQ ID NO: 48). Thesesequences can also be further varied by addition, substitution, deletionor mutation.

HIV Gag, Pol, and Integrase sequences: The HIV sequences in the Helperplasmid can be constructed from different HIV strains or clades. Forexample, HIV Gag (SEQ ID NO: 69);

HIV Pol (SEQ ID NO: 70); and HIV Int (SEQ ID NO: 71) from the Bal straincan be interchanged with the gag, pol, and int sequences contained inthe helper/helper plus Rev plasmids as outlined herein. These sequencescan also be further varied by addition, substitution, deletion ormutation.

Envelope: The VSV-G glycoprotein can be substituted with membraneglycoproteins from feline endogenous virus (RD114) (SEQ ID NO: 72),gibbon ape leukemia virus (GALV) (SEQ ID NO: 73), Rabies (FUG) (SEQ IDNO: 74), lymphocytic choriomeningitis virus (LCMV) (SEQ ID NO: 75),influenza A fowl plague virus (FPV) (SEQ ID NO: 76), Ross Riveralphavirus (RRV) (SEQ ID NO: 77), murine leukemia virus 10A1 (MLV) (SEQID NO: 78), or Ebola virus (EboV) (SEQ ID NO: 79). Sequences for theseenvelopes are identified in the sequence portion herein. Further, thesesequences can also be further varied by addition, substitution, deletionor mutation.

In summary, the 3-vector versus 4-vector systems can be compared andcontrasted, in part, as follows. The 3-vector lentiviral vector systemcontains: 1. Helper plasmid: HIV Gag, Pol, Integrase, and Rev/Tat; 2.Envelope plasmid: VSV-G/FUG envelope; and 3. Therapeutic vector: RSV5′LTR, Psi Packaging Signal, Gag fragment, RRE, Env fragment, cPPT,WPRE, and 3′delta LTR. The 4-vector lentiviral vector systemcontains: 1. Helper plasmid: HIV Gag, Pol, and Integrase; 2. Revplasmid: Rev; 3. Envelope plasmid: VSV-G/FUG envelope; and 4.Therapeutic vector: RSV 5′LTR, Psi Packaging Signal, Gag fragment, RRE,Env fragment, cPPT, WPRE, and 3′delta LTR. Sequences corresponding withthe above elements are identified in the sequence listings portionherein.

Example 2: Development of an Anti-HIV Lentivirus Vector

The purpose of this example was to develop an anti-HIV lentivirusvector.

Inhibitory RNA Designs.

The sequence of Homo sapiens chemokine C-C motif receptor 5 (CCR5)(GC03P046377) mRNA was used to search for potential siRNA or shRNAcandidates to knockdown CCR5 levels in human cells. Potential RNAinterference sequences were chosen from candidates selected by siRNA orshRNA design programs such as from the Broad Institute or the BLOCK-iTRNAi Designer from Thermo Scientific. Individual selected shRNAsequences were inserted into lentiviral vectors immediately 3′ to a RNApolymerase III promoter such as H1, U6, or 7SK to regulate shRNAexpression. These lentivirus-shRNA constructs were used to transducecells and measure the change in specific mRNA levels. The shRNA mostpotent for reducing mRNA levels were embedded individually within amicroRNA backbone to allow for expression by either the CMV or EF-1alphaRNA polymerase II promoters. The microRNA backbone was selected frommirbase.org. RNA sequences were also synthesized as synthetic siRNAoligonucleotides and introduced directly into cells without using alentiviral vector.

The genomic sequence of Bal strain of human immunodeficiency virus type1 (HIV-1 85US_BaL, accession number AY713409) was used to search forpotential siRNA or shRNA candidates to knockdown HIV replication levelsin human cells. Based on sequence homology and experience, the searchfocused on regions of the Tat and Vif genes of HIV although anindividual of skill in the art will understand that use of these regionsis non-limiting and other potential targets might be selected.Importantly, highly conserved regions of gag or pol genes could not betargeted by shRNA because these same sequences were present in thepackaging system complementation plasmids needed for vectormanufacturing. As with the CCR5 (NM 000579.3, NM 001100168.1-specific)RNAs, potential HIV-specific RNA interference sequences were chosen fromcandidates selected by siRNA or shRNA design programs such as from theGene-E Software Suite hosted by the Broad Institute(broadinstitute.org/mai/public) or the BLOCK-iT RNAi Designer fromThermo Scientific(rnadesigner.thermofisher.com/maiexpress/setOption.do?designOption=shrna&pid=6712627360706061801). Individual selected shRNA sequences were inserted intolentiviral vectors immediately 3′ to a RNA polymerase III promoter suchas H1, U6, or 7SK to regulate shRNA expression. These lentivirus-shRNAconstructs were used to transduce cells and measure the change inspecific mRNA levels. The shRNA most potent for reducing mRNA levelswere embedded individually within a microRNA backbone to allow forexpression by either the CMV or EF-1alpha RNA polymerase II promoters.

Vector Constructions.

For CCR5, Tat or Vif shRNA, oligonucleotide sequences containing BamHIand EcoRI restriction sites were synthesized by Eurofins MWG Operon,LLC. Overlapping sense and antisense oligonucleotide sequences weremixed and annealed during cooling from 70 degrees Celsius to roomtemperature. The lentiviral vector was digested with the restrictionenzymes BamHI and EcoRI for one hour at 37 degrees Celsius. The digestedlentiviral vector was purified by agarose gel electrophoresis andextracted from the gel using a DNA gel extraction kit from Invitrogen.The DNA concentrations were determined and vector to oligo (3:1 ratio)were mixed, allowed to anneal, and ligated. The ligation reaction wasperformed with T4 DNA ligase for 30 minutes at room temperature. 2.5microliters of the ligation mix were added to 25 microliters of STBL3competent bacterial cells. Transformation was achieved after heat-shockat 42 degrees Celsius. Bacterial cells were spread on agar platescontaining ampicillin and drug-resistant colonies (indicating thepresence of ampicillin-resistance plasmids) were recovered, purified andexpanded in LB broth. To check for insertion of the oligo sequences,plasmid DNA were extracted from harvested bacteria cultures with theInvitrogen DNA mini prep kit. Insertion of the shRNA sequence in thelentiviral vector was verified by DNA sequencing using a specific primerfor the promoter used to regulate shRNA expression. Exemplary vectorsequences that were determined to restrict HIV replication can be foundin FIG. 7. For example, the shRNA sequences with the highest activityagainst CCR5, Tat or Vif gene expression were then assembled into amicroRNA (miR) cluster under control of the EF-1 alpha promoter. Thepromoter and miR sequences are depicted in FIG. 7.

Further, and using standard molecular biology techniques (e.g.,Sambrook; Molecular Cloning: A Laboratory Manual, 4^(th) Ed.) as well asthe techniques described herein, a series of lentiviral vectors havebeen developed as depicted in FIG. 8 herein.

Vector 1 was developed and contains, from left to right: a long terminalrepeat (LTR) portion (SEQ ID NO: 35); a H1 element (SEQ ID NO: 101); ashCCR5 (SEQ ID NOS: 16, 18, 20, 22, or 24-Y); a posttranscriptionalregulatory element of woodchuck hepatitis virus (WPRE) (SEQ ID NOS: 32,80); and a long terminal repeat portion (SEQ ID NO: 102).

Vector 2 was developed and contains, from left to right: a long terminalrepeat (LTR) portion (SEQ ID NO: 35); a H1 element (SEQ ID NO: 101); ashRev/Tat (SEQ ID NO: 10); a H1 element (SEQ ID NO: 101); a shCCR5 (SEQID NOS: 16, 18, 20, 22, or 24); a posttranscriptional regulatory elementof woodchuck hepatitis virus (WPRE) (SEQ ID NOS: 32, 80); and a longterminal repeat portion (SEQ ID NO: 102).

Vector 3 was developed and contains, from left to right: a long terminalrepeat (LTR) portion (SEQ ID NO: 35); a H1 element (SEQ ID NO: 101); ashGag (SEQ ID NO: 12); a H1 element (SEQ ID NO: 101); a shCCR5 (SEQ IDNOS: 16, 18, 20, 22, or 24); a posttranscriptional regulatory element ofwoodchuck hepatitis virus (WPRE) (SEQ ID NOS: 32, 80); and a longterminal repeat portion (SEQ ID NO: 102).

Vector 4 was developed and contains, from left to right: a long terminalrepeat (LTR) portion (SEQ ID NO: 35); a 7SK element (SEQ ID NO: 103); ashRev/Tat (SEQ ID NO: 10); a H1 element (SEQ ID NO: 101); a shCCR5 (SEQID NOS: 16, 18, 20, 22, or 24); a posttranscriptional regulatory elementof woodchuck hepatitis virus (WPRE) (SEQ ID NOS: 32, 80); and a longterminal repeat portion (SEQ ID NO: 102).

Vector 5 was developed and contains, from left to right: a long terminalrepeat (LTR) portion (SEQ ID NO: 35); a EF1 element (SEQ ID NO: 4);miR30CCR5 (SEQ ID NO: 1); MiR21Vif (SEQ ID NO: 2); miR185Tat (SEQ ID NO:3); a posttranscriptional regulatory element of woodchuck hepatitisvirus (WPRE) (SEQ ID NOS: 32, 80); and a long terminal repeat portion(SEQ ID NO: 102).

Vector 6 was developed and contains, from left to right: a long terminalrepeat (LTR) portion (SEQ ID NO: 35); a EF1 element (SEQ ID NO: 4);miR30CCR5 (SEQ ID NO: 1); MiR21Vif (SEQ ID NO: 2); miR155Tat (SEQ ID NO:104); a posttranscriptional regulatory element of woodchuck hepatitisvirus (WPRE) (SEQ ID NOS: 32, 80); and a long terminal repeat portion(SEQ ID NO: 102).

Vector 7 was developed and contains, from left to right: a long terminalrepeat (LTR) portion (SEQ ID NO: 35); a EF1 element (SEQ ID NO: 4);miR30CCR5 (SEQ ID NO: 1); MiR21Vif (SEQ ID NO: 2); miR185Tat (SEQ ID NO:3); a posttranscriptional regulatory element of woodchuck hepatitisvirus (WPRE) (SEQ ID NOS: 32, 80); and a long terminal repeat portion(SEQ ID NO: 102).

Vector 8 was developed and contains, from left to right: a long terminalrepeat (LTR) portion (SEQ ID NO: 35); a EF1 element (SEQ ID NO: 4);miR30CCR5 (SEQ ID NO: 1); MiR21Vif (SEQ ID NO: 2); miR185Tat (SEQ ID NO:3); and a long terminal repeat portion (SEQ ID NO: 102).

Vector 9 was developed and contains, from left to right: a long terminalrepeat (LTR) portion (SEQ ID NO: 35); a CD4 element (SEQ ID NO: 30);miR30CCR5 (SEQ ID NO: 1); miR21Vif (SEQ ID NO: 2); miR185Tat (SEQ ID NO:3); a posttranscriptional regulatory element of woodchuck hepatitisvirus (WPRE) (SEQ ID NOS: 32, 80); and a long terminal repeat portion(SEQ ID NO: 102).

Development of Vectors

It should be noted that not all vectors developed for these experimentsnecessarily worked as might be predicted. More specifically, alentivirus vector against HIV might include three main components: 1)inhibitory RNA to reduce the level of HIV binding proteins (receptors)on the target cell surface to block initial virus attachment andpenetration; 2) overexpression of the HIV TAR sequence that willsequester viral Tat protein and decrease its ability to transactivateviral gene expression; and 3) inhibitory RNA that attack important andconserved sequences within the HIV genome.

With respect to the first point above, a key cell surface HIV bindingprotein is the chemokine receptor CCR5. HIV particles attach tosusceptible T cells by binding to the CD4 and CCR5 cell surfaceproteins. Because CD4 is an essential glycoprotein on the cell surfacethat is important for the immunological function of T cells, this wasnot chosen as a target to manipulate its expression levels. However,people born homozygous for null mutations in the CCR5 gene andcompletely lacking receptor expression, live normal lives save forenhanced susceptibility to a few infectious diseases and the possibilityof developing rare autoimmunity. Thus, modulating CCR5 was determined tobe a relatively safe approach and was a primary target in thedevelopment of anti-HIV lentivirus vectors.

With respect to the second point above, the viral TAR sequence is ahighly structured region of HIV genomic RNA that binds tightly to viralTat protein. The Tat:TAR complex is important for efficient generationof viral RNA. Over-expression of the TAR region was envisioned as adecoy molecule that would sequester Tat protein and decrease the levelsof viral RNA. However, TAR proved toxic to most mammalian cellsincluding cells used for manufacturing lentivirus particles. Further,TAR was inefficient for inhibiting viral gene expression in otherlaboratories and has been discarded as a viable component in HIV genetherapy.

In various embodiments, viral gene sequences have been identified thatmeet 3 criteria: i) Sequences that are reasonably conserved across arange of HIV isolates representative of the epidemic in a geographicregion of interest; ii) reduction in RNA levels due to the activity ofan inhibitory RNA in a viral vector will reduce the correspondingprotein levels by an amount sufficient to meaningfully reduce HIVreplication; and iii) the viral gene sequence(s) targeted by inhibitoryRNA are not present in the genes required for packaging and assemblingviral vector particles during manufacturing. In various embodiments, asequence at the junction of HIV Tat and Rev genes and a second sequencewithin the HIV Vif gene have been targeted by inhibitory RNA. TheTat/Rev targeting has an additional benefit of reducing HIV envelopeglycoprotein expression because this region overlaps with the envelopegene in the HIV genome.

Various methods for vector development and testing relies first onidentifying suitable targets (as described herein) followed byconstructing plasmid DNAs expressing individual or multiple inhibitoryRNA species for testing in cell models, and finally constructinglentivirus vectors containing inhibitory RNA with proven anti-HIVfunction. The lentivirus vectors are tested for toxicity, yield duringin vitro production, and effectiveness against HIV in terms of reducingCCR5 expression levels or lowering viral gene products to inhibit virusreplication.

Table 2 below demonstrates progression through multiple versions ofinhibitory constructs until arriving at a clinical candidate. Initially,shRNA (short homology RNA) molecules were designed and expressed fromplasmid DNA constructs.

Plasmids 1-4, as detailed in Table 2 below, tested shRNA sequencesagainst Gag, Pol and RT genes of HIV. While each shRNA was active forsuppressing viral protein expression in a cell model, there were twoimportant problems that prevented further development. First, thesequences were targeted to a laboratory isolate of HIV that was notrepresentative of Clade B HIV strains currently circulating in NorthAmerica and Europe. Second, these shRNA targeted critical components inthe lentivirus vector packaging system and would severely reduce vectoryield during manufacturing. Plasmid 5, as detailed in Table 2, wasselected to target CCR5 and provided a lead candidate sequence. Plasmids6, 7, 8, 9, 10, and 11, as detailed in Table 2, incorporated the TARsequence and it was found they produced unacceptable toxicity formammalian cells including cells used for lentivirus vectormanufacturing. Plasmid 2, as detailed in Table 2, identified a leadshRNA sequence capable of reducing Tat RNA expression. Plasmid 12, asdetailed in Table 2, demonstrated the effectiveness of shCCR5 expressedas a microRNA (miR) in a lentiviral vector and confirmed it should be inthe final product. Plasmid 13, as detailed in Table 2, demonstrated theeffectiveness of a shVif expressed as a microRNA (miR) in a lentiviralvector and confirmed it should be in the final product. Plasmid 14, asdetailed in Table 2, demonstrated the effectiveness of shTat expressedas a microRNA (miR) in a lentiviral vector and confirmed it should be inthe final product. Plasmid 15, as detailed in Table 2, contained the miRCCR5, miR Tat and miR Vif in the form of a miR cluster expressed from asingle promoter. These miR do not target critical components in thelentivirus vector packaging system and proved to have negligibletoxicity for mammalian cells. The miRs within the cluster were equallyeffective to individual miR that were tested previously, and the overallimpact was a substantial reduction in replication of a CCR5-tropic HIVBaL strain.

TABLE 2 Development of HIV Vectors Internal Code Material DescriptionRemarks Decision 1 SIH-H1- Lentiviral shRNA Wrong target, lab AbandonshRT-1,3 vector construct for virus, no virus test RT of LAI strain 2SIH-H1- Lentiviral H1 promoter Tat protein knock- Lead shRT43 vectorshRNA down >90% (Tat/Rev Tat/Rev NL4-3) overlapVector Construction: For Rev/Tat (RT) shRNA, oligonucleotide sequences containing BamHIand EcoRI restriction sites were synthesized by MWG Operon. Two different Rev/Tat targetsequences were tested for their ability to decrease Tat mRNA expression. The RT1,3 targetsequence is (5′-ATGGCAGGAAGAAGCGGAG-3′)(SEQ ID NO: 89) and shRNA sequenceis (5′-ATGGCAGGAAGAAGCGGAGTTCAAGAGACTCCGCTTCTTCCTGCCATTTTTT-3′)(SEQ ID NO: 90). The RT43 sequence is (5′-GCGGAGACAGCGACGAAGAGC-3′)(SEQ ID NO: 9) and shRNA sequence is (5′-GCGGAGACAGCGACGAAGAGCTTCAAGAGAGCTCTTCGTCGCTGTCTCCGCTTTTT-3′)(SEQ ID NO: 10). Oligonucleotide sequences were inserted into the pSIH lentiviral vector(System Biosciences).Functional test for shRNA against Rev/Tat: The ability of the vector to reduce Tat expressionwas tested using a luciferase reporter plasmid which contained the Rev/Tat target sequencesinserted into the 3′-UTR (untranslated region of the mRNA). Either the shRT1,3 or shRT43plasmid was co-transfected with the plasmid containing luciferase and the Rev/Tar targetsequence. There was a 90% reduction in light emission indicating strong function of theshRT43 shRNA sequence but less than 10% with the shRT1,3 plasmid.Conclusion: The SIH-H1-shRT43 was superior to SIH-H1-shRT-1,3 in terms of reducingmRNA levels in the Luciferase assay system. This indicates potent inhibitory activity of theshRT43 sequence and it was selected as a lead candidate for further development.3 SIH-H1- Lentiviral shRNA Inhibits Gag Abandon shGag-1 vectorconstruct for expression but will LAI Gag inhibit packagingVector Construction: For Gag shRNA, oligonucleotide sequences containing BamHI andEcoRI restriction sites were synthesized by MWG Operon. A Gag target sequence was testedfor their ability to decrease Gag mRNA expression. The Gag target sequence is (5′-GAAGAAATGATGACAGCAT-3′)(SEQ ID NO: 11) and shRNA sequence is (5′-GAAGAAATGATGACAGCATTTCAAGAGAATGCTGTCATCATTTCTTCTTTTT-3′)(SEQ ID NO: 12). Oligonucleotide sequences were inserted into the pSIH lentiviral vector(System Biosciences).Functional test for shRNA against Gag: The ability of the vector to reduce Gag expression wastested using a luciferase reporter plasmid which contained the Gag target sequences insertedinto the 3′-UTR (untranslated region of the mRNA). The Gag plasmid was co-transfectedwith the plasmid containing luciferase and the Gag target sequence. There was nearly a 90%reduction in light emission indicating a strong effect of the shGag shRNA sequence.Conclusion: This shRNA sequence is potent against HIV Gag expression but was abandoned.The lentivirus packaging system requires production of Gag from the helper plasmid andshRNA inhibition of Gag will reduce lentivirus vector yield. This shRNA sequence could beused as an oligonucleotide inhibitor of HIV or incorporated into an alternate viral vectorpackaging system that uses a different vector genome or is modified to resist inhibition by thisshRNA. 4 SIH-H1- Lentiviral shRNA Inhibits Pol Abandon shPol-1 vectorconstruct for expression but will Pol inhibit packagingVector Construction: A Pol shRNA was constructed with oligonucleotide sequences containingBamHI and EcoRI restriction sites that were synthesized by MWG Operon. A Pol targetsequence was tested for its ability to decrease Pol mRNA expression. The Pol target sequenceis (5′-CAGGAGCAGATGATACAG-3′)(SEQ ID NO: 13) and shRNA sequence is (5′-CAGGAGATGATACAGTTCAAGAGACTGTATCATCTGCTCCTGTTTTT-3′)(SEQ IDNO: 14). Oligonucleotide sequences were inserted into the pSIH lentiviral vector (SystemBiosciences).Functional tests for shRNA against HIV Pot: The ability of the vector to reduce Pol expressionwas tested using a luciferase reporter plasmid which contained the Pol target sequencesinserted into the 3′-UTR (untranslated region of the mRNA). The Pol plasmid was co-transfected with the plasmid containing luciferase and the Pol target sequence. There was a60% reduction in light emission indicating a strong effect of the shPol shRNA sequence.Conclusion: This shRNA sequence is potent against HIV Pol expression but was abandoned.The lentivirus packaging system requires production of Pol from the helper plasmid andshRNA inhibition of Pol will reduce lentivirus vector yield. This shRNA sequence could beused as an oligonucleotide inhibitor of HIV or incorporated into an alternate viral vectorpackaging system that uses a different vector genome or is modified to resist inhibition by thisshRNA. 5 SIH-H1- Lentiviral shRNA Best of 5 Lead shCCR5-1 vectorconstruct for candidates, CCR5 Extracellular CCR5 protein reduction >90%Vector Construction: A CCR5 shRNA was constructed with oligonucleotide sequencescontaining BamHI and EcoRI restriction sites that were synthesized by MWG Operon.Oligonucleotide sequences were inserted into the pSIH lentiviral vector (System Biosciences).The CCR5 target sequence #1, which focuses on CCR5 gene sequence 1 (SEQ ID NO: 25), is(5′-GTGTCAAGTCCAATCTATG-3′)(SEQ ID NO: 15) and the shRNA sequence is (5′-GTGTCAAGTCCAATCTATGTTCAAGAGACATAGATTGGACTTGACACTTTTT-3′)(SEQ ID NO: 16). The CCR5 target sequence #2, which focuses on CCR5 gene sequence 2(SEQ ID NO: 26), is (5′-GAGCATGACTGACATCTAC-3′)(SEQ ID NO: 17) and theshRNA sequence is (5′-GAGCATGACTGACATCTACTTCAAGAGAGTAGATGTCAGTCATGCTCTTTTT-3′)(SEQ ID NO: 18). The CCR5 target sequence #3, which focuses on CCR5 gene sequence 3(SEQ ID NO: 27), is (5′-GTAGCTCTAACAGGTTGGA-3′)(SEQ ID NO: 19) and theshRNA sequence is (5′-GTAGCTCTAACAGGTTGGATTCAAGAGATCCAACCTGTTAGAGCTACTTTTT-3′)(SEQ ID NO: 20). The CCR5 target sequence #4, which focuses on CCR5 gene sequence 4(SEQ ID NO: 28, is (5′-GTTCAGAAACTACCTCTTA-3′)(SEQ ID NO: 21) and the shRNAsequence is (5′-GTTCAGAAACTACCTCTTATTCAAGAGATAAGAGGTAGTTTCTGAACTTTTT-3′)(SEQ ID NO: 22). The CCR5 target sequence #5, which focuses on CCR5 gene sequence 5(SEQ ID NO: 29), is (5′-GAGCAAGCTCAGTTTACACC-3′)(SEQ ID NO: 23) and theshRNA sequence is (5′-GAGCAAGCTCAGTTTACACCTTCAAGAGAGGTGTAAACTGAGCTTGCTCTTTTT-3′)(SEQ ID NO: 24).Functional test for shRNA against CCR5: The ability of a CCR5 shRNA sequence to knock-down CCR5 RNA expression was initially tested by co-transfecting each of the lentiviralplasmids, in separate experiments for each plasmid, containing one of the five CCR5 targetsequences with a plasmid expressing the human CCR5 gene. CCR5 mRNA expression wasthen assessed by qPCR analysis using CCR5-specific primers.Conclusion: Based on the reduction in CCR5 mRNA levels the shRNACCR5-1 was mostpotent for reducing CCR5 gene expression. This shRNA was selected as a lead candidate.6 SIH-U6- Lentiviral U6 promoter- Toxic to cells Abandon TAR vector TAR7 SIH-U6- Lentiviral U6 promoter- Toxic to cells Abandon TAR-H1- vectorTAR-H1- shCCR5 shCCR5 8 U6-TAR- Lentiviral U6 promoter- Suppress HIV,Abandon H1-shRT vector TAR-H1-RT toxic to cells, poor packaging 9U6-TAR- Lentiviral Change shRNA Toxic, poor Abandon 7SK-shRT vectorpromoter to packaging 7SK 10 U6-TAR- Lentiviral U6 promoter- Toxic, poorAbandon H1-shRT- vector TAR-H1-RT- packaging, H1 H1-shCCR5 H1-shCCR5repeats 11 U6-TAR- Lentiviral Change shRNA Toxic, poor Abandon 7SK-shRT-vector promoter to packaging H1-CCR5 7SKVector Construction: A TAR decoy sequence containing flanking KpnI restriction sites wassynthesized by MWG operon and inserted into the pSIH lentiviral vector (System Biosciences)at the KpnI site. In this vector, TAR expression is regulated by the U6 promoter. The TARdecoy sequence is (5′-CTTGCAATGATGTCGTAATTTGCGTCTTACCTCGTTCTCGACAGCGACCAGATCTGAGCCTGGGAGCTCTCTGGCTGTCAGTAAGCTGGTACAGAAGGTTGACGAAAATTCTTACTGAGCAAGAAA-3′)(SEQ ID NO: 8). Expression of the TAR decoy sequence wasdetermined by qPCR analysis using specific primers for the TAR sequence. Additional vectorswere constructed also containing the TAR sequence. The H1 promoter and shRT sequencewas inserted in this vector in the XhoI site. The H1 shRT sequence is (5′-GAACGCTGACGTCATCAACCCGCTCCAAGGAATCGCGGGCCCAGTGTCACTAGGCGGGAACACCCAGCGCGCGTGCGCCCTGGCAGGAAGATGGCTGTGAGGGACAGGGGAGTGGCGCCCTGCAATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGACCACTTGGATCCGCGGAGACAGCGACGAAGAGCTTCAAGAGAGCTCTTCGTCGCTGTCTCCGCTTTTT-3′)(SEQ ID NO: 91). This vector could express TAR and knockdown RT. The 7SKpromoter was also substituted for the H1 promoter to regulate shRT expression. Anothervector was constructed containing U6 TAR, H1 shRT, and H1 shCCR5. The H1 shCCR5sequence was inserted into the SpeI site of the plasmid containing U6 TAR and H1 shRT. TheH1 CCR5 sequence is (5′-GAACGCTGACGTCATCAACCCGCTCCAAGGAATCGCGGGCCCAGTGTCACTAGGCGGGAACACCCAGCGCGCGTGCGCCCTGGCAGGAAGATGGCTGTGAGGGACAGGGGAGTGGCGCCCTGCAATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGACCACTTGGATCCGTGTCAAGTCCAATCTATGTTCAAGAGACATAGATTGGACTTGACACTTTTT-3′)(SEQ ID NO: 92). The 7SK promoter was also substituted for the H1 promoter to regulateshRT expression.Functional test for TAR decoy activity: We tested the effect of SIH-U6-TAR on packagingefficiency. When TAR sequence was included, the yield of vector in the SIH packaging systemwas reduced substantially.Conclusion: Lentivirus vectors expressing the TAR decoy sequence are unsuitable forcommercial development due to low vector yields. These constructs were abandoned.12 shCCR5 Lentiviral microRNA Extracellular CCR5 Lead vector sequenceprotein reduction >90%Vector Construction: A CCR5 microRNA was constructed with oligonucleotide sequencescontaining BsrGI and NotI restriction sites that were synthesized by MWG Operon.Oligonucleotide sequences were inserted into the pCDH lentiviral vector (SystemBiosciences). The EF-1 promoter was substituted for a CMV promoter that was used in theplasmid construct Test Material 5. The EF-1 promoter was synthesized by MWG Operoncontaining flanking ClaI and BsrGI restriction sites and inserted into the pCDH vectorcontaining shCCR5-1. The EF-1 promoter sequence is (5′-CCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACGCCCCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA-3′)(SEQ ID NO: 4).Functional test for lentivirus CDH-shCCR5-1: The ability of the miR CCR5 sequences toknock-down CCR5 expression was determined by transducing CEM-CCR5 T cells andmeasuring cell surface CCR5 expression after staining with a fluorescently-labeled monoclonalantibody against CCR5 and measuring the intensity of staining, that is directly proportional tothe number of cell surface CCR5 molecules, by analytical flow cytometry. The most effectiveshRNA sequence for targeting CCR5 was CCR5 shRNA sequence #1. However, the mosteffective CCR5 targeting sequence for constructing the synthetic microRNA sequence wasoverlapping with CCR5 sequence #5; this conclusion was based on sequence alignments andexperience with miRNA construction. Finally, the miR30 hairpin sequence was used toconstruct the synthetic miR30 CCR5 sequence which is (5′-AGGTATATTGCTGTTGACAGTGAGCGACTGTAAACTGAGCTTGCTCTACTGTGAAGCCACAGATGGGTAGAGCAAGCACAGTTTACCGCTGCCTACTGCCTCGGACTTCAAGGGGCTT-3′)(SEQ ID NO: 1). The miR CCR5 target sequence is (5′-GAGCAAGCTCAGTTTACA-3′)(SEQ ID NO: 5). At multiplicity of infection equal to 5,generating on average 1.25 genome copies of integrated lentivirus per cell, CCR5 expressionlevels were reduce by ≥90% indicating potent inhibition of CCR5 mRNA by the miR30CCR5micro RNA construct in a lentivirus vector.Conclusion: The miR30CCR5 construct is potent for reducing CCR5 cell surface expressionand is a lead candidate for a therapeutic lentivirus for HIV. 13 shVifLentiviral microRNA Vif protein Lead vector sequence reduction >80%Vector Construction: A Vif microRNA was constructed with oligonucleotide sequencescontaining BsrGI and NotI restriction sites that were synthesized by MWG Operon.Oligonucleotide sequences were inserted into the pCDH lentiviral vector (System Biosciences)containing an EF-1 promoter. Based on sequence alignments and experience with constructingsynthetic miRNA, the miR21 hairpin sequence was used to construct the synthetic miR21 Vifsequence which is (5′-CATCTCCATGGCTGTACCACCTTGTCGGGGGATGTGTACTTCTGAACTTGTGTTGAATCTCATGGAGTTCAGAAGAACACATCCGCACTGACATTTTGGTATCTTTCATCTGACCA-3′)(SEQ ID NO: 2). The miR Vif target sequence is (5′-GGGATGTGTACTTCTGAACTT-3′)(SEQ ID NO: 6).Functional test for potency of miR21Vif: The ability of the miR Vif sequence to knock-downVif expression was determined by measuring Vif protein expression by immunoblot analysisusing an anti-Vif monoclonal antibody to identify the Vif protein.Conclusion: the miR21Vif reduced Vif protein expression by ≥10-fold as determined byquantitative image analysis of immunoblot data. This was sufficient to justify miR21Vif as alead candidate for our therapeutic lentivirus. 14 shTat LentiviralmicroRNA Tat RNA Lead vector sequence reduction >80%Vector Construction: A Tat microRNA was constructed with oligonucleotide sequencescontaining BsrGI and NotI restriction sites that were synthesized by MWG Operon. ThemicroRNA cluster was inserted into the pCDH lentiviral vector (System Biosciences)containing an EF-1 promoter. Based on sequence alignments and experience in theconstruction of synthetic miRNA, the miR185 hairpin sequence was selected for constructing asynthetic miR185 Tat sequence which is (5′-GGGCCTGGCTCGAGCAGGGGGCGAGGGATTCCGCTTCTTCCTGCCATAGCGTGGTCCCCTCCCCTATGGCAGGCAGAAGCGGCACCTTCCCTCCCAATGACCGCGTCTTCGTCG-3′). The miR Tat target sequence is (5′-TCCGCTTCTTCCTGCCATAG-3′)(SEQ IDNO: 7).Functional test for potency of miR185Tat: The ability of miR Tat to knock-down Tatexpression was determined by measuring Tat mRNA expression by RT-PCR analysis usingTat specific primers. We compared the miR185Tat with a similar miR155Tat on the basis ofreducing the relative levels of Tat mRNA.Conclusion: The miR185Tat was approximately twice as potent for reducing Tat mRNAcompare to miR155Tat, and was selected as the lead candidate for our therapeutic lentivirus.15 shCCR5- Lentiviral microRNA CCR5 Candidate shVif-shTat vector clusterreduction >90%, Vif sequence protein reduction >80%, Tat RNAreduction >80%, >95% inhibition of HIV replicationVector Construction: A miR30CCR5 miR21Vif miR185Tat microRNA cluster sequence wasconstructed with a synthetic DNA fragment containing BsrGI and NotI restriction sites thatwas synthesized by MWG Operon. The DNA fragment was inserted into the pCDH lentiviralvector (System Biosciences) containing the EF-1 promoter. The miR cluster sequence is (5′-AGGTATATTGCTGTTGACAGTGAGCGACTGTAAACTGAGCTTGCTCTACTGTGAAGCCACAGATGGGTAGAGCAAGCACAGTTTACCGCTGCCTACTGCCTCGGACTTCAAGGGGCTTCCCGGGCATCTCCATGGCTGTACCACCTTGTCGGGGGATGTGTACTTCTGAACTTGTGTTGAATCTCATGGAGTTCAGAAGAACACATCCGCACTGACATTTTGGTATCTTTCATCTGACCAGCTAGCGGGCCTGGCTCGAGCAGGGGGCGAGGGATTCCGCTTCTTCCTGCCATAGCGTGGTCCCCTCCCCTATGGCAGGCAGAAGCGGCACCTTCCCTCCCAATGACCGCGTCTTCGTC-3′)(SEQ ID NO: 31) and incorporates Test Material12, Test Material 13 and Test Material 14 into a single cluster that can be expressed undercontrol of the EF-1 promoter.Functional test for potency of the Lentivirus Vector AGT103 containing the microRNA clusterof miR30CCR5, miR21Vif and miR185Tat: The AGT103 vector was tested for potency againstCCR5 using the assay for reduction in cell surface CCR5 expression (Test Material 12). TheAGT103 vector was tested for potency against Vif using the assay for reduction in cell surfaceVif expression (Test Material 13). The AGT103 vector was tested for potency against Tatusing the assay for reduction in cell surface Tat expression (Test Material 14).Conclusion: Potency for reducing CCR5 expression by the miRNA cluster was similar topotency observed for the miR30CCR5 alone. Potency for reducing Vif expression by themiRNA cluster was similar to potency observed for the miR21Vif alone. Potency for reducingTat expression by the miRNA cluster was similar to potency observed for the miR185Tatalone. The miRNA cluster is potent for reducing cell surface CCR5 levels and for inhibitingtwo HIV genes. Thus, AGT103 containing this miRNA cluster was selected as the therapeuticvector construct for our HIV functional cure program.

Functional Assays.

Individual lentivirus vectors containing CCR5, Tat or Vif shRNAsequences and, for experimental purposes, expressing green fluorescentprotein (GFP) under control of the CMV Immediate Early Promoter, anddesignated AGT103/CMV-GFP were tested for their ability to knockdownCCR5, Tat or Vif expression. Mammalian cells were transduced withlentiviral particles either in the presence or absence of polybrene.Cells were collected after 2-4 days; protein and RNA were analyzed forCCR5, Tat or Vif expression. Protein levels were tested by Western blotassay or by labeling cells with specific fluorescent antibodies (CCR5assay), followed by analytical flow cytometry comparing modified andunmodified cell fluorescence using either the CCR5-specific or isotypecontrol antibodies.

Starting Testing of Lentivirus.

T cell culture medium was made using RPMI 1640 supplemented with 10% FBSand 1% penicillin—streptomycin. Cytokine stocks of IL-2 10,000 units/ml,IL-12 1 μg/ml, IL-7 1 μg/ml, IL-15 1 μg/ml were also prepared inadvance.

Prior to transduction with the lentivirus, an infectious viral titer wasdetermined and used to calculate the amount of virus to add for theproper multiplicity of infection (MOI).

Day 0-12: Antigen-Specific Enrichment.

On day 0, cryopreserved PBMC were thawed, washed with 10 ml 37° C.medium at 1200 rpm for 10 minutes and resuspended at a concentration of2×10⁶/ml in 37° C. medium. The cells were cultured at 0.5 ml/well in a24-well plate at 3TC in 5% CO2. To define the optimal stimulationconditions, cells were stimulated with combinations of reagents aslisted in Table 3 below:

TABLE 3 1 2 3 4 5 6 IL-2 + IL-12 IL-7 + IL-15 Peptides + Peptides +MVA + IL- MVA + IL- IL-2 + IL-12 IL-7 + IL-15 2 + IL-12 7 + IL-15

Final concentrations: IL-2=20 units/ml, IL-12=10 ng/ml, IL-7=10 ng/ml,IL-15=10 ng/ml, peptides=5 μg/ml individual peptide, MVA MOI=1.

On days 4 and 8, 0.5 ml fresh medium and cytokine at listedconcentrations (all concentrations indicate the final concentration inthe culture) were added to the stimulated cells.

Day 12-24: Non-Specific Expansion and Lentivirus Transduction.

On day 12, the stimulated cells were removed from the plate by pipettingand resuspended in fresh T cell culture medium at a concentration of1×10⁶/ml. The resuspended cells were transferred to T25 culture flasksand stimulated with DYNABEADS® Human T-Activator CD3/CD28 following themanufacturer's instruction plus cytokine as listed above; flasks wereincubated in the vertical position.

On day 14, AGT103/CMV-GFP was added at MOI 20 and cultures were returnedto the incubator for 2 days. At this time, cells were recovered bypipetting, collected by centrifugation at 1300 rpm for 10 minutes,resuspended in the same volume of fresh medium, and centrifuged again toform a loose cell pellet. That cell pellet was resuspended in freshmedium with the same cytokines used in previous steps, with cells at0.5×10⁶ viable cells per ml.

From days 14 to 23, the number of the cells was evaluated every 2 daysand the cells were diluted to 0.5×10⁶/ml with fresh media. Cytokineswere added every time.

On day 24, the cells were collected and the beads were removed from thecells. To remove the beads, cells were transferred to a suitable tubethat was placed in the sorting magnet for 2 minutes. Supernatantcontaining the cells was transferred to a new tube. Cells were thencultured for 1 day in fresh medium at 1×10⁶/ml. Assays were performed todetermine the frequencies of antigen-specific T cells and lentivirustransduced cells.

To prevent possible viral outgrowth, amprenavir (0.5 ng/ml) was added tothe cultures on the first day of stimulation and every other day duringthe culture.

Examine Antigen-Specific T Cells by Intracellular Cytokine Staining forIFN-Gamma.

Cultured cells after peptide stimulation or after lentivirustransduction at 1×10⁶ cells/ml were stimulated with medium alone(negative control), Gag peptides (5 μg/ml individual peptide), or PHA (5μg/ml, positive control). After 4 hours, BD GolgiPlug™ (1:1000, BDBiosciences) was added to block Golgi transport. After 8 hours, cellswere washed and stained with extracellular (CD3, CD4 or CD8; BDBiosciences) and intracellular (IFN-gamma; BD Biosciences) antibodieswith BD Cytofix/Cytoperm™ kit following the manufacturer's instruction.Samples were analyzed on a BD FACSCalibur™ Flow Cytometer. Controlsamples labeled with appropriate isotype-matched antibodies wereincluded in each experiment. Data were analyzed using Flowjo software.

Lentivirus transduction rate was determined by the frequency of GFP+cells. The transduced antigen-specific T cells are determined by thefrequency of CD3+CD4+GFP+IFN gamma+cells; tests for CD3+CD8+GFP+IFNgamma+cells are included as a control.

These results indicate that CD4 T cells, the target T cell population,can be transduced with lentiviruses that are designed to specificallyknock down the expression of HIV-specific proteins, thus producing anexpandable population of T cells that are immune to the virus. Thisexample serves as a proof of concept indicating that the disclosedlentiviral constructs can be used in combination with vaccination toproduce a functional cure in HIV patients.

Example 4: CCR5 Knockdown with Experimental Vectors

AGTc120 is a Hela cell line that stably expresses large amounts of CD4and CCR5. AGTc120 was transduced with or without LV-CMV-mCherry (the redfluorescent protein mCherry expressed under control of the CMV ImmediateEarly Promoter) or AGT103/CMV-mCherry. Gene expression of the mCherryfluorescent protein was controlled by a CMV (cytomegalovirus immediateearly promoter) expression cassette. The LV-CMV-mCherry vector lacked amicroRNA cluster, while AGT103/CMV-mCherry expressed therapeutic miRNAagainst CCR5, Vif, and Tat.

As shown in FIG. 9A, transduction efficiency was >90%. After 7 days,cells were collected and stained with fluorescent monoclonal antibodyagainst CCR5 and subjected to analytical flow cytometry. Isotypecontrols are shown in gray on these histograms plotting MeanFluorescence Intensity of CCR5 APC (x axis) versus cell numbernormalized to mode (y axis). After staining for cell surface CCR5, cellstreated with no lentivirus or control lentivirus (expressing only themCherry marker) showed no changes in CCR5 density while AGT103 (rightsection) reduced CCR5 staining intensity to nearly the levels of isotypecontrol. After 7 days, cells were infected with or without R5-tropic HIVreporter virus Bal-GFP. 3 days later, cells were collected and analyzedby flow cytometry. More than 90% of cells were transduced.AGT103-CMV/CMVmCherry reduced CCR5 expression in transduced AGTc120cells and blocked R5-tropic HIV infection compared with cells treatedwith the Control vector.

FIG. 9B shows the relative insensitivity of transfected AGTc120 cells toinfection with HIV. As above, the lentivirus vectors express mCherryprotein and a transduced cell that was also infected with HIV(expressing GFP) would appear as a double positive cell in the upperright quadrant of the false color flow cytometry dot plots. In theabsence of HIV (upper panels), there were no GFP+ cells under anycondition. After HIV infection (lower panels), 56% of cells wereinfected in the absence of lentivirus transduction and 53.6% of cellsbecame infected in AGTc120 cells transduced with the LV-CMV-mCherry.When cells were transduced with the therapeutic AGT103/CMV-mCherryvector, only 0.83% of cells appeared in the double positive quadrantindicating they were transduced and infected.

Dividing 53.62 (proportion of double positive cells with control vector)by 0.83 (the proportion of double positive cells with the therapeuticvector) shows that AGT103 provided greater than 65-fold protectionagainst HIV in this experimental system.

Example 5: Regulation of CCR5 Expression by shRNA Inhibitor Sequences ina Lentiviral Vector

Inhibitory RNA Design.

The sequence of Homo sapiens chemokine receptor CCR5 (CCR5, NC000003.12) was used to search for potential siRNA or shRNA candidates toknockdown CCR5 levels in human cells. Potential RNA interferencesequences were chosen from candidates selected by siRNA or shRNA designprograms such as from the Broad Institute or the BLOCK-IT RNA iDesignerfrom Thermo Scientific. A shRNA sequence may be inserted into a plasmidimmediately after a RNA polymerase III promoter such as H1, U6, or 7SKto regulate shRNA expression. The shRNA sequence may also be insertedinto a lentiviral vector using similar promoters or embedded within amicroRNA backbone to allow for expression by an RNA polymerase IIpromoter such as CMV or EF-1 alpha. The RNA sequence may also besynthesized as a siRNA oligonucleotide and utilized independently of aplasmid or lentiviral vector.

Plasmid Construction.

For CCR5 shRNA, oligonucleotide sequences containing BamHI and EcoRIrestriction sites were synthesized by MWG Operon. Oligonucleotidesequences were annealed by incubating at 70° C. then cooled to roomtemperature. Annealed oligonucleotides were digested with therestriction enzymes BamHI and EcoRI for one hour at 37° C., then theenzymes were inactivated at 70° C. for 20 minutes. In parallel, plasmidDNA was digested with the restriction enzymes BamHI and EcoRI for onehour at 37° C. The digested plasmid DNA was purified by agarose gelelectrophoresis and extracted from the gel using a DNA gel extractionkit from Invitrogen. The DNA concentration was determined and the plasmato oligonucleotide sequence was ligated in the ratio 3:1 insert tovector. The ligation reaction was done with T4 DNA ligase for 30 minutesat room temperature. 2.5 μL of the ligation mix were added to 25 μL ofSTBL3 competent bacterial cells. Transformation required heat shock at42° C. Bacterial cells were spread on agar plates containing ampicillinand colonies were expanded in L broth. To check for insertion of theoligo sequences, plasmid DNA was extracted from harvested bacterialcultures using the Invitrogen DNA Miniprep kit and tested by restrictionenzyme digestion. Insertion of the shRNA sequence into the plasmid wasverified by DNA sequencing using a primer specific for the promoter usedto regulate shRNA expression.

Functional Assay for CCR5 mRNA Reduction:

The assay for inhibition of CCR5 expression required co-transfection oftwo plasmids. The first plasmid contains one of five different shRNAsequences directed against CCR5 mRNA. The second plasmid contains thecDNA sequence for human CCR5 gene. Plasmids were co-transfected into293T cells. After 48 hours, cells were lysed and RNA was extracted usingthe RNeasy kit from Qiagen. cDNA was synthesized from RNA using a SuperScript Kit from Invitrogen. The samples were then analyzed byquantitative RT-PCR using an Applied Biosystems Step One PCR machine.CCR5 expression was detected with SYBR Green from Invitrogen using theforward primer (5′-AGGAATTGATGGCGAGAAGG-3′) (SEQ ID NO: 93) and reverseprimer (5′-CCCCAAAGAAGGTCAAGGTAATCA-3′) (SEQ ID NO: 94) with standardconditions for polymerase chain reaction analysis. The samples werenormalized to the mRNA for beta actin gene expression using the forwardprimer (5′-AGCGCGGCTACAGCTTCA-3′) (SEQ ID NO: 95) and reverse primer(5′-GGCGACGTAGCACAGCTTCT-3′) (SEQ ID NO: 96) with standard conditionsfor polymerase chain reaction analysis. The relative expression of CCR5mRNA was determined by its Ct value normalized to the level of actinmessenger RNA for each sample. The results are shown in FIG. 10.

As shown in FIG. 10A, CCR5 knock-down was tested in 293T cells byco-transfection of the CCR5 shRNA construct and a CCR5-expressingplasmid. Control samples were transfected with a scrambled shRNAsequence that did not target any human gene and the CCR5-expressingplasmid. After 60 hours post-transfection, samples were harvested andCCR5 mRNA levels were measured by quantitative PCR. Further, as shown inFIG. 10B, CCR5 knock-down after transduction with lentivirus expressingCCR5 shRNA-1 (SEQ ID NO: 16).

Example 6: Regulation of HIV Components by shRNA Inhibitor Sequences ina Lentiviral Vector

Inhibitory RNA Design.

The sequences of HIV type 1 Rev/Tat (5′-GCGGAGACAGCGACGAAGAGC-3′) (SEQID NO: 9) and Gag (5′-GAAGAAATGATGACAGCAT-3′) (SEQ ID NO: 11) were usedto design:

Rev/Tat: (5′GCGGAGACAGCGACGAAGAGCTTCAAGAGAGCTCTTCGTCGCTGTCTCCGCTTTTT-3′) (SEQ ID NO: 10) and Gag:

(5′GAAGAAATGATGACAGCATTTCAAGAGAATGCTGTCATCATTTCTTCTTTTT-3′) (SEQ ID NO:12) shRNA that were synthesized and cloned into plasmids as describedabove.

Plasmid Construction.

The Rev/Tat or Gag target sequences were inserted into the 3′UTR(untranslated region) of the firefly luciferase gene used commonly as areporter of gene expression in cells or tissues. Additionally, oneplasmid was constructed to express the Rev/Tat shRNA and a secondplasmid was constructed to express the Gag shRNA. Plasmid constructionswere as described above.

Functional Assay for shRNA Targeting of Rev/Tat or Gag mRNA:

Using plasmid co-transfection we tested whether a shRNA plasmid wascapable of degrading luciferase messenger RNA and decreasing theintensity of light emission in co-transfected cells. A shRNA control(scrambled sequence) was used to establish the maximum yield of lightfrom luciferase transfected cells. When the luciferase constructcontaining a Rev/Tat target sequence inserted into the 3′-UTR(untranslated region of the mRNA) was co-transfected with the Rev/TatshRNA sequence there was nearly a 90% reduction in light emissionindicating strong function of the shRNA sequence. A similar result wasobtained when a luciferase construct containing a Gag target sequence inthe 3′-UTR was co-transfected with the Gag shRNA sequence. These resultsindicate potent activity of the shRNA sequences.

As shown in FIG. 11A, knock-down of the Rev/Tat target gene was measuredby a reduction of luciferase activity, which was fused with the targetmRNA sequence in the 3′UTR, by transient transfection in 293T cells. Asshown in FIG. 11B, knock-down of the Gag target gene sequence fused withthe luciferase gene. The results are displayed as the mean±SD of threeindependent transfection experiments, each in triplicate.

Example 7: AGT103 Decreases Expression of Tat and Vif

Cells were transfected with exemplary vector AGT103/CMV-GFP. AGT103 andother exemplary vectors are defined in Table 3 below.

TABLE 3 Vector Designation Composition AGT103EF1-miR30CCR5-miR21Vif-miR185-Tat-WPRE Control-mCherry CMV-mCherryAGT103/CMV- CMV-mCherry-EF1-miR30CCR5-miR21Vif- mCherry miR185-Tat-WPRE-Control-GFP CMV-mCherry AGT103/CMV-GFP CMV-GFP-EF1-miR30CCR5-miR21Vif-miR185-Tat-WPRE- Abbreviations: EF-1: elongation factor 1transcriptional promoter miR30CCR5 - synthetic microRNA capable ofreducing CCR5 protein on cell surfaces miR21Vif - synthetic microRNAcapable of reducing levels of HIV RNA and Vif protein expressionmiR185Tat - synthetic micro RNA capable of reducing levels of HIV RNAand Tat protein expression CMV - Immediate early transcriptionalpromoter from human cytomegalovirus mCherry - coding region for themCherry red fluorescent protein GFP - coding region for the greenfluorescent protein WPRE - Woodchuck hepatitis virus posttranscriptional regulatory element

A T lymphoblastoid cell line (CEM; CCRF-CEM; American Type CultureCollection Catalogue number CCL119) was transduced with AGT103/CMV-GFP.48 hours later the cells were transfected with an HIV expression plasmidencoding the entire viral sequence. After 24 hours, RNA was extractedfrom cells and tested for levels of intact Tat sequences using reversetranscriptase polymerase chain reaction. Relative expression levels forintact Tat RNA were reduced from approximately 850 in the presence ofcontrol lentivirus vector, to approximately 200 in the presence ofAGT103/CMV-GFP for a total reduction of >4 fold, as shown in FIG. 12.

Example 8: Regulation of HIV Components by Synthetic MicroRNA Sequencesin a Lentiviral Vector

Inhibitory RNA Design.

The sequence of HIV-1 Tat and Vif genes were used to search forpotential siRNA or shRNA candidates to knockdown Tat or Vif levels inhuman cells. Potential RNA interference sequences were chosen fromcandidates selected by siRNA or shRNA design programs such as from theBroad Institute or the BLOCK-IT RNA iDesigner from Thermo Scientific.The selected shRNA sequences most potent for Tat or Vif knockdown wereembedded within a microRNA backbone to allow for expression by an RNApolymerase II promoter such as CMV or EF-I alpha. The RNA sequence mayalso be synthesized as a siRNA oligonucleotide and used independently ofa plasmid or lentiviral vector.

Plasmid Construction.

The Tat target sequence (5′-TCCGCTTCTTCCTGCCATAG-3′) (SEQ ID NO: 7) wasincorporated into the miR185 backbone to create a Tat miRNA(5′-GGGCCTGGCTCGAGCAGGGGGCGAGGGATTCCGCTTCTTCCTGCCATAGCGTGGTCCCCTCCCCTATGGCAGGCAGAAGCGGCACCTTCCCTCCCAATGACCGCGTCTTCG TCG-3′) (SEQ IDNO: 3) that was inserted into a lentivirus vector and expressed undercontrol of the EF-1 alpha promoter. Similarly, the Vif target sequence(5′-GGGATGTGTACTTCTGAACTT-3′) (SEQ ID NO: 6) was incorporated into themiR21 backbone to create a Vif miRNA(5′-CATCTCCATGGCTGTACCACCTTGTCGGGGGATGTGTACTTCTGAACTTGTGTTGAATCTCATGGAGTTCAGAAGAACACATCCGCACTGACATTTTGGTATCTTTCATCTG ACCA-3′) (SEQID NO: 2) that was inserted into a lentivirus vector and expressed undercontrol of the EF-1 alpha promoter. The resulting Vif/TatmiRNA-expressing lentivirus vectors were produced in 293T cells using alentiviral vector packaging system. The Vif and Tat miRNA were embeddedinto a microRNA cluster consisting of miR CCR5, miR Vif, and miR Tat allexpressed under control of the EF-1 promoter.

Functional Assay for miR185Tat Inhibition of Tat mRNA Accumulation.

A lentivirus vector expressing miR185 Tat (LV-EF1-miR—CCR5-Vif-Tat) wasused at a multiplicity of infection equal to 5 for transducing 293Tcells. 24 hours after transduction the cells were transfected with aplasmid expressing HIV strain NL4-3 (pNL4-3) using Lipofectamine2000under standard conditions. 24 hours later RNA was extracted and levelsof Tat messenger RNA were tested by RT-PCR using Tat-specific primersand compared to actin mRNA levels for a control.

Functional Assay for miR21 Vif Inhibition of Vif Protein Accumulation.

A lentivirus vector expressing miR21 Vif (LV-EF1-miR—CCR5-Vif-Tat) wasused at a multiplicity of infection equal to 5 for transducing 293Tcells. 24 hours after transduction, the cells were transfected with aplasmid expressing HIV strain NL4-3 (pNL4-3) using Lipofectamine2000. 24hours later cells were lysed and total soluble protein was tested tomeasure the content of Vif protein. Cell lysates were separated bySDS-PAGE according to established techniques. The separated proteinswere transferred to nylon membranes and probed with a Vif-specificmonoclonal antibody or actin control antibody.

As shown in FIG. 13A, Tat knock-down was tested in 293T cells transducedwith either a control lentiviral vector or a lentiviral vectorexpressing either synthetic miR185 Tat or miR155 Tat microRNA. After 24hours, the HIV vector pNL4-3 was transfected with Lipofectamine2000 for24 hours and then RNA was extracted for qPCR analysis with primers forTat. As shown in FIG. 13B, Vif knock-down was tested in 293T cellstransduced with either a control lentiviral vector or a lentiviralvector expressing a synthetic miR21 Vif microRNA. After 24 hours, theHIV vector pNL4-3 was transfected with Lipofectamine2000 for 24 hoursand then protein was extracted for immunoblot analysis with an antibodyfor HIV Vif.

Example 9: Regulation of CCR5 Expression by Synthetic microRNA Sequencesin a Lentiviral Vector

CEM-CCR5 cells were transduced with a lentiviral vector containing asynthetic miR30 sequence for CCR5 (AGT103: TGTAAACTGAGCTTGCTCTA (SEQ IDNO: 97), AGT103-R5-1: TGTAAACTGAGCTTGCTCGC (SEQ ID NO: 98), orAGT103-R5-2: CATAGATTGGACTTGACAC (SEQ ID NO: 99). After 6 days, CCR5expression was determined by FACS analysis with an APC-conjugated CCR5antibody and quantified by mean fluorescence intensity (MFI). CCR5levels were expressed as % CCR5 with LV-Control set at 100%. The targetsequence of AGT103 and AGT103-R5-1 is in the same region as CCR5 targetsequence #5. The target sequence of AGT103-R5-2 is the same as CCR5target sequence #1. AGT103 (2% of total CCR5) is most effective atreducing CCR5 levels as compared with AGT103-R5-1 (39% of total CCR5)and AGT103-R5-2 which does not reduce CCR5 levels. The data isdemonstrated in FIG. 14 herein.

Example 10: Regulation of CCR5 Expression by Synthetic microRNASequences in a Lentiviral Vector Containing Either a Long or Short WPRESequence

Vector Construction.

Lentivirus vectors often require an RNA regulatory element for optimalexpression of therapeutic genes or genetic constructs. A common choiceis to use the Woodchuck hepatitis virus post transcriptional regulatoryelement (WPRE). We compared AGT103 that contains a full-length WPRE:

(SEQ ID NO: 32) (5′AATCAACCTCTGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCT-3′)with a modified AGT103 vector containing a shortened WPRE element

(SEQ ID NO: 80) (5′AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTAATGCCTCTGTATCATGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTC-3′).

Functional Assay for Modulating Cell Surface CCR5 Expression as aFunction of Long Versus Short WPRE Element in the Vector Sequence.

AGT103 containing long or short WPRE elements were used for transducingCEM-CCR5 T cells a multiplicity of infection equal to 5. Six days aftertransduction cells were collected and stained with a monoclonal antibodycapable of detecting cell surface CCR5 protein. The antibody wasconjugated to a fluorescent marker and the intensity of staining isdirectly proportional to the level of CCR5 on the cell surface. Acontrol lentivirus had no effect on cell surface CCR5 levels resultingin a single population with a mean fluorescence intensity of 73.6 units.The conventional AGT103 with a long WPRE element reduced CCR5 expressionto a mean fluorescence intensity level of 11 units. AGT103 modified toincorporate a short WPRE element resulted in a single population ofcells with mean fluorescence intensity of 13 units. Accordingly,substituting a short WPRE element had little or no effect on thecapacity for AGT103 to reduce cell surface CCR5 expression.

As shown in FIG. 14, CEM-CCR5 cells were transduced with AGT103containing either a long or short WPRE sequence. After 6 days, CCR5expression was determined by FACS analysis with an APC-conjugated CCR5antibody and quantified as mean fluorescence intensity (MFI). CCR5levels were expressed as % CCR5 with LV-Control set at 100%. Thereduction in CCR5 levels was similar for AGT103 with either the short(5.5% of total CCR5) or long (2.3% of total CCR5) WPRE sequence.

Example 11: Regulation of CCR5 Expression by Synthetic microRNASequences in a Lentiviral Vector with or without a WPRE Sequence

Vector Construction.

In order to test whether WPRE was required for AGT103 down regulation ofCCR5 expression we constructed a modified vector without WPRE elementsequences.

Functional assay for modulating cell surface CCR5 expression as afunction of including or not including a long WPRE element in the AGT103vector. In order to test whether WPRE was required for AGT103 modulationof CCR5 expression levels we transduced CEM-CCR5 T cells with AGT103 ora modified vector lacking WPRE using a multiplicity of infection equalto 5. Six days after transduction cells were collected and stained witha monoclonal antibody capable of recognizing cell surface CCR5 protein.The monoclonal antibody was directly conjugated to a fluorescent markerand the intensity of staining is directly proportional to the number ofCCR5 molecules per cell surface. A lentivirus control vector had noeffect on cell surface CCR5 levels resulting in a uniform populationwith mean fluorescence intensity of 164. The lentivirus vector (AGT103with a long WPRE and also expressing GFP marker protein), AGT103 lackingGFP but containing a long WPRE element, or AGT103 lacking both GFP andWPRE all were similarly effective for modulating cell surface CCR5expression. After removing GFP, AGT103 with or without WPRE elementswere indistinguishable in terms of their capacity for modulating cellsurface CCR5 expression.

CEM-CCR5 cells were transduced with AGT103 with or without GFP and WPRE.After 6 days, CCR5 expression was determined by FACS analysis with anAPC-conjugated CCR5 antibody and quantified as mean fluorescenceintensity (MFI). CCR5 levels were expressed as % CCR5 with LV-Controlset at 100%. The reduction in CCR5 levels was similar for AGT103 with(0% of total CCR5) or without (0% of total CCR5) the WPRE sequence. Thisdata is demonstrated in FIG. 16.

Example 12: Regulation of CCR5 Expression by a CD4 Promoter RegulatingSynthetic microRNA Sequences in a Lentiviral Vector

Vector Construction.

A modified version of AGT103 was constructed to test the effect ofsubstituting alternate promoters for expressing the microRNA clusterthat suppresses CCR5, Vif and Tat gene expression. In place of thenormal EF-1 promoter we substituted the T cell-specific promoter for CD4glycoprotein expression using the sequence:

(SEQ ID NO: 30) (5′TGTTGGGGTTCAAATTTGAGCCCCAGCTGTTAGCCCTCTGCAAAGAAAAAAAAAAAAAAAAAAGAACAAAGGGCCTAGATTTCCCTTCTGAGCCCCACCCTAAGATGAAGCCTCTTCTTTCAAGGGAGTGGGGTTGGGGTGGAGGCGGATCCTGTCAGCTTTGCTCTCTCTGTGGCTGGCAGTTTCTCCAAAGGGTAACAGGTGTCAGCTGGCTGAGCCTAGGCTGAACCCTGAGACATGCTACCTCTGTCTTCTCATGGCTGGAGGCAGCCTTTGTAAGTCACAGAAAGTAGCTGAGGGGCTCTGGAAAAAAGACAGCCAGGGTGGAGGTAGATTGGTCTTTGACTCCTGATTTAAGCCTGATTCTGCTTAACTTTTTCCCTTGACTTTGGCATTTTCACTTTGACATGTTCCCTGAGAGCCTGGGGGGTGGGGAACCCAGCTCCAGCTGGTGACGTTTGGGGCCGGCCCAGGCCTAGGGTGTGGAGGAGCCTTGCCATCGGGCTTCCTGTCTCTCTTCATTTAAGCACGACTCTGCAGA-3′).

Functional Assay Comparing EF-1 and CD4 Gene Promoters in Terms ofPotency for Reducing Cell Surface CCR5 Protein Expression.

AGT103 modified by substituting the CD4 gene promoter for the normalEF-1 promoter was used for transducing CEM-CCR5 T cells. Six days aftertransduction cells were collected and stained with a monoclonal antibodycapable of recognizing cell surface CCR5 protein. The monoclonalantibody was conjugated to a fluorescent marker and staining intensityis directly proportional to the level of cell surface CCR5 protein. Acontrol lentivirus transduction resulted in a population of CEM-CCR5 Tcells that were stained with a CCR5-specific monoclonal antibody andproduced a mean fluorescence intensity of 81.7 units. The modifiedAGT103 using a CD4 gene promoter in place of the EF-1 promoter forexpressing microRNA showed a broad distribution of staining with a meanfluorescence intensity roughly equal to 17.3 units. Based on thisresult, the EF-1 promoter is at least similar and likely superior to theCD4 gene promoter for microRNA expression. Depending on the desiredtarget cell population, the EF-1 promoter is universally active in allcell types and the CD4 promoter is only active in T-lymphocytes.

CEM-CCR5 cells were transduced with a lentiviral vector containing a CD4promoter regulating a synthetic microRNA sequence for CCR5, Vif, and Tat(AGT103). After 6 days, CCR5 expression was determined by FACS analysiswith an APC-conjugated CCR5 antibody and quantified as mean fluorescenceintensity (MFI). CCR5 levels were expressed as % CCR5 with LV-Controlset at 100%. In cells transduced with LV-CD4-AGT103, CCR5 levels were11% of total CCR5. This is comparable to that observed for LV-AGT103which contains the EF1 promoter. This data is demonstrated in FIG. 17.

Example 13: Detecting HIV Gag-Specific CD4 T Cells

Cells and Reagents.

Viable frozen peripheral blood mononuclear cells (PBMC) were obtainedfrom a vaccine company. Data were obtained with a representativespecimen from an HIV+ individual who was enrolled into an early stageclinical trial (TRIAL REGISTRATION: clinicaltrials.gov NCT01378156)testing a candidate HIV therapeutic vaccine. Two specimens were obtainedfor the “Before vaccination” and “After vaccination” studies. Cellculture products, supplements and cytokines were from commercialsuppliers. Cells were tested for responses to recombinant ModifiedVaccinia Ankara 62B from Geovax Corporation as described in Thompson etal. (2016). “DNA/MVA Vaccination of HIV-1 Infected Participants withViral Suppression on Antiretroviral Therapy, followed by TreatmentInterruption: Elicitation of Immune Responses without Control ofRe-Emergent Virus.” PLoS One 11(10): e0163164. Synthetic peptidesrepresenting the entire HIV-1 Gag polyprotein were obtained from GeoVaxthe HIV (GAG) Ultra peptide sets were obtained from JPT PeptideTechnologies GmbH (www.jpt.com), Berlin, Germany. HIV (GAG) Ultracontains 150 peptides each being 15 amino acids in length andoverlapping by 11 amino acids. They were chemically synthesized thenpurified and analyzed by liquid chromatography—mass spectrometry.Collectively these peptides represent major immunogenic regions of theHIV Gag polyprotein and are designed for average coverage of 57.8% amongknown HIV strains. Peptide sequences are based on the HIV sequencedatabase from the Los Alamos National Laboratory(http://www.hiv.lanl.gov/content/sequence/NEWALIGN/align.html). Peptidesare provided as dried trifluoroacetate salts, 25 micrograms per peptide,and are dissolved in approximately 40 microliters of DMSO then dilutedwith PBS to final concentration. Monoclonal antibodies for detecting CD4and cytoplasmic IFN-gamma were obtained from commercial sources andintracellular staining was done with the BD Pharmingen IntracellularStaining Kit for interferon-gamma. Peptides were resuspended in DMSO andwe include a DMSO only control condition.

Functional Assay for Detecting HIV-Specific CD4+ T Cells.

Frozen PBMC were thawed, washed and resuspended in RPMI mediumcontaining 10% fetal bovine serum, supplements and cytokines. CulturedPBMC collected before or after vaccination were treated with DMSOcontrol, MVA GeoVax (multiplicity of infection equal to 1 plaque formingunit per cell), Peptides GeoVax (1 microgram/ml) or HIV (GAG) Ultrapeptide mixture (1 microgram/ml) for 20 hours in the presence of GolgiStop reagent. Cells were collected, washed, fixed, permeabilized andstained with monoclonal antibodies specific for cell surface CD4 orintracellular interferon-gamma. Stained cells were analyzed with aFACSCalibur analytical flow cytometer and data were gated on the CD4+ Tcell subset. Cells highlighted within boxed regions are double-positiveand designated HIV-specific CD4 T cells on the basis of interferon-gammaexpression after MVA or peptide stimulation. Numbers within the boxedregions show the percentage of total CD4 that were identified asHIV-specific. We did not detect strong responses to DMSO or MVA.Peptides from GeoVax elicited fewer responding cells compared to HIV(GAG) Ultra peptide mixture from JPT but differences were small and notsignificant.

As shown in FIG. 18, PBMCs from a HIV-positive patient before or aftervaccination were stimulated with DMSO (control), recombinant MVAexpressing HIV Gag from GeoVax (MVA GeoVax), Gag peptide from GeoVax(Pep GeoVax, also referred to herein as Gag peptide pool 1) or Gagpeptides from JPT (HIV (GAG) Ultra, also referred to herein as Gagpeptide pool 2) for 20 hours. IFNg production was detected byintracellular staining and flow cytometry using standard protocols. Flowcytometry data were gated on CD4 T cells. Numbers captured in boxes arethe percentage of total CD4 T cells designated “HIV-specific” on thebasis of cytokine response to antigen-specific stimulation.

Example 14: HIV-Specific CD4 T Cell Expansion and LentivirusTransduction

Designing and testing methods for enriching PBMC to increase theproportion of HIV-specific CD4 T cells and transducing these cells withAGT103 to produce the cellular product AGT103T.

The protocol was designed for ex vivo culture of PBMC (peripheral bloodmononuclear cells) from HIV-positive patients who had received atherapeutic HIV vaccine. In this example, the therapeutic vaccineconsisted of three doses of plasmid DNA expressing HIV Gag, Pol and Envgenes followed by two doses of MVA 62-B (modified vaccinia Ankara number62-B) expressing the same HIV Gag, Pol, and Env genes. The protocol isnot specific for a vaccine product and only requires a sufficient levelof HIV-specific CD4+ T cells after immunization. Venous blood wascollected and PBMC were purified by Ficoll-Paque density gradientcentrifugation. Alternately, PBMC or defined cellular tractions can beprepared by positive or negative selection methods using antibodycocktails and fluorescence activated or magnetic bead sorting. Thepurified PBMC are washed and cultured in standard medium containingsupplements, antibiotics and fetal bovine serum. To these cultures, apool of synthetic peptides was added representing possible T cellepitopes within the HIV Gag polyprotein. Cultures are supplemented byadding cytokines interleukin-2 and interleukin-12 that were selectedafter testing combinations of interleukin-2 and interleukin-12,interleukin 2 and interleukin-7, interleukin 2 and interleukin-15.Peptide stimulation is followed by a culture interval of approximately12 days. During the 12 days culture, fresh medium and fresh cytokinesupplements were added approximately once every four days.

The peptide stimulation interval is designed to increase the frequencyof HIV-specific CD4 T cells in the PBMC culture. These HIV-specific CD4T cells were activated by prior therapeutic immunization and can bere-stimulated and caused to proliferate by synthetic peptide exposure.Our goal is to achieve greater than or equal to 1% of total CD4 T cellsbeing HIV-specific by end of the peptide stimulation culture period.

On approximately day 12 of culture cells are washed to remove residualmaterials then stimulated with synthetic beads decorated with antibodiesagainst CD4 T cell surface proteins CD3 and CD28. This well-establishedmethod for polyclonal stimulation of T cells will reactivate the cellsand make them more susceptible for AGT103 lentivirus transduction. Thelentivirus transduction is performed on approximately day 13 of cultureand uses a multiplicity of infection between 1 and 5. After transductioncells are washed to remove residual lentivirus vector and cultured inmedia containing interleukin-2 and interleukin-12 with fresh medium andcytokines added approximately once every four days until approximatelyday 24 of culture.

Throughout the culture interval the antiretroviral drug Saquinavir isadded at a concentration of approximately 100 nM to suppress anypossible outgrowth of HIV.

On approximately day 24 of culture cells are harvested, washed, a sampleis set aside for potency and release assay, then the remaining cells aresuspended in cryopreservation medium before freezing in single aliquotsof approximately 1×10¹⁰ cells per dose that will contain approximately1×10⁸ HIV-specific CD4 T cells that are transduced with AGT103.

Potency of the cell product (AGT103T) is tested in one of two alternatepotency assays. Potency assay 1 tests for the average number of genomecopies (integrated AGT103 vector sequences) per CD4 T cell. The minimumpotency is approximately 0.5 genome copies per CD4 T cell in order torelease the product. The assay is performed by positive selection of CD3positive/CD4 positive T cells using magnetic bead labeled monoclonalantibodies, extracting total cellular DNA and using a quantitative PCRreaction to detect sequences unique to the AGT103 vector. Potency assay2 tests for the average number of genome copies of integrated AGT103within the subpopulation of HIV-specific CD4 T cells. This essay isaccomplished by first stimulating the PBMC with the pool of syntheticpeptides representing HIV Gag protein. Cells are then stained with aspecific antibody reagent capable of binding to the CD4 T cell and alsocapturing secreted interferon-gamma cytokine. The CD4positive/interferon-gamma positive cells are captured by magnetic beadselection, total cellular DNA is prepared, and the number of genomecopies of AGT103 per cell is determined with a quantitative PCRreaction. Release criterion based on potency using Assay 2 require thatgreater than or equal to 0.5 genome copies per HIV-specific CD4 T-cellare present in the AGT103 cell product.

Functional Test for Enriching and Transducing HIV-Specific CD4 T Cellsfrom PBMC of HIV-Positive Patients that Received a Therapeutic HIVVaccine.

The impact of therapeutic vaccination on the frequency of HIV-specificCD4 T cells was tested by a peptide stimulation assay (FIG. 19, PanelB). Before vaccination the frequency of HIV-specific CD4 T cells was0.036% in this representative individual. After vaccination, thefrequency of HIV-specific CD4 T cells was increased approximately 2-foldto the value of 0.076%. Responding cells (HIV-specific) identified byaccumulation of cytoplasmic interferon-gamma, were only detected afterspecific peptide stimulation.

We also tested whether peptide stimulation to enrich for HIV-specificCD4 T cells followed by AGT103 transduction would reach our goal ofgenerating approximately 1% of total CD4 T cells in culture that wereboth HIV-specific and transduced by AGT103. In this case, we used anexperimental version of AGT103 that expresses green fluorescence protein(see GFP). In FIG. 19, Panel C the post-vaccination culture afterpeptide stimulation (HIV (GAG) Ultra) and AGT103 transductiondemonstrated that 1.11% of total CD4 T cells were both HIV-specific(based on expressing interferon-gamma in response to peptidestimulation) and AGT103 transduced (based on expression of GFP).

Several patients from a therapeutic HIV vaccine study were tested toassess the range of responses to peptide stimulation and to begindefining eligibility criteria for entering a gene therapy arm in afuture human clinical trial. FIG. 19 Panel D show the frequency ofHIV-specific CD4 T cells in 4 vaccine trial participants comparing theirpre- and post-vaccination specimens. In three cases the post-vaccinationspecimens show a value of HIV-specific CD4 T cells that was greater thanor equal to 0.076% of total CD4 T cells. The ability to reach this valuewas not predicted by the pre-vaccination specimens as patient 001-004and patient 001-006 both started with pre-vaccination values of 0.02%HIV-specific CD4 T cells but one reached an eventual post-vaccinationvalue of 0.12% HIV-specific CD4 T cells while the other individual failto increase this value after vaccination. The same three patients thatresponded well to vaccine, in terms of increasing the frequency ofHIV-specific CD4 T cells, also showed substantial enrichment ofHIV-specific CD4 T cells after peptide stimulation and culture. In thethree cases shown in FIG. 19 Panel E, peptide stimulation and subsequentculture generated samples where 2.07%, 0.72% or 1.54% respectively oftotal CD4 T cells were HIV-specific. These values indicate that amajority of individuals responding to a therapeutic HIV vaccine willhave a sufficiently large ex vivo response to peptide stimulation inorder to enable our goal of achieving approximately 1% of total CD4 Tcells that are HIV-specific and transduced with AGT103 in the final cellproduct.

As shown in FIG. 19, Panel A describes the schedule of treatment. PanelB demonstrates that PBMCs were stimulated with Gag peptide or DMSOcontrol for 20 hours. IFN gamma production was detected by intracellularstaining by FACS. CD4⁺ T cells were gated for analysis. Panel Cdemonstrates CD4⁺ T cells were expanded and transduced with AGT103-GFPusing the method as shown in Panel A. Expanded CD4⁺ T cells were restedin fresh medium without any cytokine for 2 days and re-stimulated withGag peptide or DMSO control for 20 hours. IFN gamma production and GFPexpression was detected by FACS. CD4⁺ T cells were gated for analysis.Panel D demonstrates frequency of HIV-specific CD4⁺ T cells (IFN gammapositive, pre- and post-vaccination) were detected from 4 patients.Panel E demonstrates Post-vaccination PBMCs from 4 patients wereexpanded and HIV-specific CD4⁺ T cells were examined.

Example 15: Dose Response

Vector Construction.

A modified version of AGT103 was constructed to test the dose responsefor increasing AGT103 and its effects on cell surface CCR5 levels. TheAGT103 was modified to include a green fluorescent protein (GFP)expression cassette under control of the CMV promoter. Transduced cellsexpression the miR30CCR5 miR21Vif miR185Tat micro RNA cluster and emitgreen light due to expressing GFP.

Functional Assay for Dose Response of Increasing AGT103-GFP andInhibition of CCR5 Expression.

CEM-CCR5 T cells were transduced with AGT103-GFP using multiplicity ofinfection per cell from 0 to 5. Transduced cells were stained with afluorescently conjugated (APC) monoclonal antibody specific for cellsurface CCR5. The intensity of staining is proportional to the number ofCCR5 molecules per cell surface. The intensity of green fluorescence isproportional to the number of integrated AGT103-GFP copies per cell.

As shown in FIG. 20, Panel A demonstrates the dose response forincreasing AGT103-GFP and its effects on cell surface CCR5 expression.At multiplicity of infection equal to 0.4 only 1.04% of cells are bothgreen (indicating transduction) and showing significantly reduced CCR5expression. At multiplicity of infection equal to 1 the number ofCCR5low, GFP+ cells increases to 68.1%/ At multiplicity of infectionequal to 5 the number of CCR5low, GFP+ cells increased to 95.7%. Thesedata are presented in histogram form in FIG. 20, Panel B that shows anormally distribution population in terms of CCR5 staining, movingtoward lower mean fluorescence intensity with increasing doses ofAGT103-GFP. The potency of AGT103-GFP is presented in graphical form inFIG. 20, Panel C showing the percentage inhibition of CCR5 expressionwith increasing doses of AGT103-GFP. At multiplicity of infection equalto 5, there was greater than 99% reduction in CCR5 expression levels.

Example 16: AGT103 Efficiently Transduces Primary Human CD4⁺ T Cells

Transducing Primary CD4 T Cells with AGT103 Lentivirus Vector.

A modified AGT103 vector containing the green fluorescence proteinmarker (GFP) was used at multiplicities of infection between 0.2 and 5for transducing purified, primary human CD4 T cells.

Functional Assay for Transduction Efficiency of AGT103 in Primary HumanCD4 T Cells.

CD4 T cells were isolated from human PBMC (HIV-negative donor) usingmagnetic bead labeled antibodies and standard procedures. The purifiedCD4 T cells were stimulated ex vivo with CD3/CD28 beads and cultured inmedia containing interleukin-2 for 1 day before AGT103 transduction. Therelationship between lentivirus vector dose (the multiplicity ofinfection) and transduction efficiency is demonstrated in FIG. 21, PanelA showing that multiplicity of infection equal to 0.2 resulted in 9.27%of CD4 positive T cells being transduced by AGT103 and that value wasincreased to 63.1% of CD4 positive T cells being transduced by AGT103with a multiplicity of infection equal to 5. In addition to achievingefficient transduction of primary CD4 positive T cells it is alsonecessary to quantify the number of genome copies per cell. In FIG. 21,Panel B total cellular DNA from primary human CD4 T cells transduced atseveral multiplicities of infection were tested by quantitative PCR todetermine the number of genome copies per cell. In a multiplicity ofinfection equal to 0.2 we measured 0.096 genome copies per cell that wasin good agreement with 9.27% GFP positive CD4 T cells in panel A.Multiplicity of infection equal to 1 generated 0.691 genome copies percell and multiplicity of infection equal to 5 generated 1.245 genomecopies per cell.

As shown in FIG. 21, CD4⁺ T cells isolated from PBMC were stimulatedwith CD3/CD28 beads plus IL-2 for 1 day and transduced with AGT103 atvarious concentrations. After 2 days, beads were removed and CD4⁺ Tcells were collected. As shown in Panel A, frequency of transduced cells(GFP positive) were detected by FACS. As shown in Panel B, the number ofvector copies per cell was determined by qPCR. At a multiplicity ofinfection (MOI) of 5, 63% of CD4⁺ T cells were transduced with anaverage of 1 vector copy per cell.

Example 17: AGT103 Inhibits HIV Replication in Primary CD4⁺ T Cells

Protecting Primary Human CD4 Positive T Cells from HIV Infection byTransducing Cells with AGT103.

Therapeutic lentivirus AGT103 was used for transducing primary human CD4positive T cells at multiplicities of infection between 0.2 and 5 percell. The transduced cells were then challenged with a CXCR4-tropic HIVstrain NL4.3 that does not require cell surface CCR5 for penetration.This assay tests the potency of microRNA against Vif and Tat genes ofHIV in terms of preventing productive infection in primary CD4 positiveT cells, but uses an indirect method to detect the amount of HIVreleased from infected, primary human CD4 T cells.

Functional Assay for AGT103 Protection Against CXCR4-Tropic HIVInfection of Primary Human CD4 Positive T Cells.

CD4 T cells were isolated from human PBMC (HIV-negative donor) usingmagnetic bead labeled antibodies and standard procedures. The purifiedCD4 T cells were stimulated ex vivo with CD3/CD28 beads and cultured inmedia containing interleukin-2 for 1 day before AGT103 transductionusing multiplicities of infection between 0.2 and 5. Two days aftertransduction the CD4 positive T cell cultures were challenged with HIVstrain NL4.3 that was engineered to express the green fluorescentprotein (GFP). The transduced and HIV-exposed primary CD4 T cellcultures were maintained for 7 days before collecting cell-free culturefluids containing HIV. The cell-free culture fluids were used to infecta highly permissive T cell line C8166 for 2 days. The proportion ofHIV-infected C8166 cells was determined by flow cytometry detecting GFPfluorescence. With a mock lentivirus infection, the dose of 0.1multiplicity of infection for NL4.3 HIV resulted in an amount of HIVbeing released into culture fluids that was capable of establishingproductive infection in 15.4% of C8166 T cells. With the dose 0.2multiplicity of infection for AGT103, this value for HIV infection ofC8166 cells is reduced to 5.3% and multiplicity of infection equal to 1for AGT103 resulted in only 3.19% of C8166 T cells being infected byHIV. C8166 infection was reduced further to 0.62% after AGT103transduction using a multiplicity of infection equal to 5. There is aclear dose response relationship between the amount of AGT103 used fortransduction and the amount of HIV released into the culture medium.

As shown in FIG. 22, CD4⁺ T cells isolated from PBMC were stimulatedwith CD3/CD28 beads plus IL-2 for 1 day and transduced with AGT103 atvarious concentrations (MOI). After 2 days, beads were removed and CD4⁺T cells were infected with 0.1 MOI of HIV NL4.3-GFP. 24 hours later,cells were washed 3 times with PBS and cultured with IL-2 (30U/ml) for 7days. At the end of the culture, supernatant was collected to infect theHIV permissive cell line C8166 for 2 days. HIV-infected C8166 cells (GFPpositive) were detected by FACS. There was a reduction in viable HIVwith an increase in the multiplicity of infection of AGT103 as observedby less infection of C8166 cells MOI 0.2=65.6%, MOI 1=79.3%, and MOI5=96%).

Example 18: AGT103 Protects Primary Human CD4⁺ T Cells from HIV-InducedDepletion

AGT103 Transduction of Primary Human CD4 T Cells to Protect AgainstHIV-Mediated Cytopathology and Cell Depletion.

PBMC were obtained from healthy, HIV-negative donors and stimulated withCD3/CD28 beads then cultured for 1 day in medium containinginterleukin-2 before AGT103 transduction using multiplicities ofinfection between 0.2 and 5.

Functional Assay for AGT103 Protection of Primary Human CD4 T CellsAgainst HIV-Mediated Cytopathology.

AGT103-transduced primary human CD4 T cells were infected with HIV NL4.3 strain (CXCR4-tropic) that does not require CCR5 for cellular entry.When using the CXCR4-tropic NL 4.3, only the effect of Vif and TatmicroRNA on HIV replication is being tested. The dose of HIV NL 4.3 was0.1 multiplicity of infection. One day after HIV infection, cells werewashed to remove residual virus and cultured in medium plusinterleukin-2. Cells were collected every three days during a 14-dayculture then stained with a monoclonal antibody that was specific forCD4 and directly conjugated to a fluorescent marker to allow measurementof the proportion of CD4 positive T cells in PBMC. Untreated CD4 T cellsor CD4 T cells transduced with the control lentivirus vector were highlysusceptible to HIV challenge and the proportion of CD4 positive T cellsin PBMC fell below 10% by day 14 culture. In contrast, there was adose-dependent effect of AGT103 on preventing cell depletion by HIVchallenge. With a AGT103 dose of 0.2 multiplicity of infection more than20% of PBMC were CD4 T cells by day 14 of culture and this valueincreased to more than 50% of PBMC being CD4 positive T cells by day 14of culture with a AGT103 dose of multiplicity of infection equal to 5.Again, there is a clear dose response effect of AGT103 on HIVcytopathogenicity in human PBMC.

As shown in FIG. 23, PBMCs were stimulated with CD3/CD28 beads plus IL-2for 1 day and transduced with AGT103 at various concentrations (MOI).After 2 days, beads were removed and cells were infected with 0.1 MOI ofHIV NL4.3. 24 hours later, cells were washed 3 times with PBS andcultured with IL-2 (30U/ml). Cells were collected every 3 days and thefrequency of CD4⁺ T cells were analyzed by FACS. After 14 days ofexposure to HIV, there was an 87% reduction in CD4⁺ T cells transducedwith LV-Control, a 60% reduction with AGT103 MOI 0.2, a 37% reductionwith AGT103 MOI 1, and a 17% reduction with AGT103 MOI 5.

Example 19: Generating a Population of CD4+ T Cells Enriched forHIV-Specificity and Transduced with AGT103/CMV-GFP

Therapeutic vaccination against HIV had minimal effect on thedistribution of CD4+, CD8+ and CD4+/CD8+ T cells. As shown in FIG. 24A,the CD4 T cell population is shown in the upper left quadrant of theanalytical flow cytometry dot plots, and changes from 52% to 57% oftotal T cells after the vaccination series. These are representativedata.

Peripheral blood mononuclear cells from a participant in an HIVtherapeutic vaccine trial were cultured for 12 days inmedium+/−interleukin-2/interleukin-12 or+/−interleukin-7/interleukin-15. Some cultures were stimulated withoverlapping peptides representing the entire p55 Gag protein of HIV-1(HIV (GAG) Ultra peptide mixture) as a source of epitope peptides for Tcell stimulation. These peptides are 10-20 amino acids in length andoverlap by 20-50% of their length to represent the entire Gag precursorprotein (p55) from HIV-1 BaL strain. The composition and sequence ofindividual peptides can be adjusted to compensate for regionalvariations in the predominant circulating HIV sequences or when detailedsequence information is available for an individual patient receivingthis therapy. At culture end, cells were recovered and stained withanti-CD4 or anti-CD8 monoclonal antibodies and the CD3+ population wasgated and displayed here. The HIV (GAG) Ultra peptide mixturestimulation for either pre- or post-vaccination samples was similar tothe medium control indicating that HIV (GAG) Ultra peptide mixture wasnot toxic to cells and was not acting as a polyclonal mitogen. Theresults of this analysis can be found in FIG. 24B.

HIV (GAG) Ultra peptide mixture and interleukin-2/interleukin-12provided for optimal expansion of antigen-specific CD4 T cells. As shownin the upper panels of FIG. 24C, there was an increase in cytokine(interferon-gamma) secreting cells in post-vaccination specimens exposedto HIV (GAG) Ultra peptide mixture. In the pre-vaccination sample,cytokine secreting cells increased from 0.43 to 0.69% as a result ofexposure to antigenic peptides. In contrast, the post-vaccinationsamples showed an increase of cytokine secreting cells from 0.62 to1.76% of total CD4 T cells as a result of peptide stimulation. Thesedata demonstrate the strong impact of vaccination on the CD4 T cellresponses to HIV antigen.

Finally, AGT103/CMV-GFP transduction of antigen-expanded CD4 T cellsproduced HIV-specific and HIV-resistant helper CD4 T cells that areneeded for infusion into patients as part of a functional cure for HIV(in accordance with other various aspects and embodiments, AGT103 aloneis used; for example, clinical embodiments may not include the CMV-GFPsegment). The upper panels of FIG. 24D show the results of analyzing theCD4+ T cell population in culture. The x axis of FIG. 24D shows GreenFluorescent Protein (GFP) emission indicating that individual cells weretransduced with the AGT103/CMV-GFP. As shown in FIG. 24D, in thepost-vaccination samples 1.11% of total CD4 T cells that were bothcytokine secreting was recovered, indicating that the cells areresponding specifically to HIV antigen, and transduced withAGT103/CMV-GFP. This is the target cell population and the clinicalproduct intended for infusion and functional cure of HIV. With theefficiency of cell expansion during the antigen stimulation andsubsequent polyclonal expansion phases of ex vivo culture, 4×10⁸antigen-specific, lentivirus transduced CD4 T cells can be produced.This exceeds the target for cell production by 4-fold and will allowachievement of a count of antigen-specific and HIV-resistant CD4 T cellsof approximately 40 cells/microliter of blood or around 5.7% of totalcirculating CD4 T cells.

Table 4 below shows the results of the ex vivo production ofHIV-specific and HIV-resistant CD4 T cells using the disclosed vectorsand methods.

TABLE 4 Percentage Percentage HIV- HIV- specific andMaterial/manipulation Total CD4 T cells specific HIV-resistantLeukapheresis pack   ~7 × 10⁸ ~0.12 N/A from HIV + patient Peptideexpansion ex   ~8 × 10⁸ ~2.4 N/A vivo Mitogen expansion ~1.5 × 10¹⁰ ~2.4N/A Lentivirus transduction ~1.5 × 10¹⁰ ~2.4 ~1.6

Example 20: Clinical Study for Treatment of HIV

AGT103T is a genetically modified autologous PBMC containing >5×10⁷HIV-specific CD4 T cells that are also transduced with AGT103 lentivirusvector.

A Phase I clinical trial will test the safety and feasibility ofinfusing ex vivo modified autologous CD4 T cells (AGT103T) in adultresearch participants with confirmed HIV infection, CD4+ T-cellcounts >600 cells per mm³ of blood and stable virus suppression below200 copies per ml of plasma while on cART. All study participants willcontinue receiving their standard antiretroviral medications through thePhase I clinical trial. Up to 40 study participants receive two doses byintramuscular injection 8 weeks apart, of recombinant modified vacciniaAnkara (rMVA) expressing HIV Gag, Pol and Env proteins. Seven to 10 daysafter the second immunization a blood sample is collected for in vitrotesting to measure the frequency of CD4+ T-cells that respond tostimulation with a pool of overlapping, synthetic peptides representingthe HIV-1 Gag polyprotein. Subjects in the upper half of vaccineresponders, based on measuring the frequency of Gag-specific CD4 T cellsare enrolled in the gene therapy arm and subjects in the lower half ofresponders do not continue in the study. We anticipate that the cut-offfor higher responders is a HIV-specific CD4+ T cell frequency ≥0.065% oftotal CD4 T cells. Subjects enrolled into the gene therapy arm of ourtrial undergo leukapheresis followed by purification of PBMC (usingFicoll density gradient centrifugation or negative selection withantibodies) that are cultured ex vivo and stimulated with HIV Gagpeptides plus interleukin-2 and interleukin-12 for 12 days, thenstimulated again with beads decorated with CD3/CD28 bispecific antibody.The antiretroviral drug Saquinavir is included at 100 nM to preventemergence of autologous HIV during ex vivo culture. One day afterCD3/CD28 stimulation cells are transduced with AGT103 at multiplicity ofinfection between 1 and 10. The transduced cells are cultured for anadditional 7-14 days during which time they expand by polyclonalproliferation. The culture period is ended by harvesting and washingcells, setting aside aliquots for potency and safety release assays, andresuspending the remaining cells in cryopreservation medium. A singledose is ≤1×10¹⁰ autologous PBMC. The potency assay measures thefrequency of CD4 T cells that respond to peptide stimulation byexpressing interferon-gamma. Other release criteria include the productmust include ≥0.5×10⁷ HIV-specific CD4 T cells that are also transducedwith AGT103. Another release criterion is that the number of AGT103genome copies per cell must not exceed 3. Five days before infusion withAGT103T subjects receive one dose of busulfuram (or Cytoxan)conditioning regimen followed by infusion of ≤1×10¹⁰ PBMC containinggenetically modified CD4 T cells.

A Phase II study will evaluate efficacy of AGT103T cell therapy. PhaseII study participants include individuals enrolled previously in ourPhase I study who were judged to have successful and stable engraftmentof genetically modified, autologous, HIV-specific CD4 T cells andclinical responses defined as positive changes in parameters monitoredas described in efficacy assessments (1.3.). Study participants will beasked to add Maraviroc to their existing regimen of antiretroviralmedication. Maraviroc is a CCR5 antagonist that will enhance theeffectiveness of genetic therapy directed at reducing CCR5 levels. Oncethe Maraviroc regimen is in place subjects will be asked to discontinuethe previous antiretroviral drug regimen and only maintain Maravirocmonotherapy for 28 days or until plasma viral RNA levels exceed 10,000per ml on 2 sequential weekly blood draws. Persistently high viremiarequires participants to return to their original antiretroviral drugregimen with or without Maraviroc according to recommendations of theirHIV care physician.

If participants remain HIV suppressed (below 2,000 vRNA copies per ml ofplasma) for >28 days on Maraviroc monotherapy, they will be asked togradually reduce Maraviroc dosing over a period of 4 weeks followed byintensive monitoring for an additional 28 days. Subjects who maintainedHIV suppression with Maraviroc monotherapy are considered to have afunctional cure. Subjects who maintain HIV suppression even afterMaraviroc withdrawal also have a functional cure. Monthly monitoring for6 months followed by less intensive monitoring will establish thedurability of functional cure.

Patient Selection

Inclusion Criteria:

-   -   Aged between 18 and 60 years.    -   Documented HIV infection prior to study entry.    -   Must be willing to comply with study-mandated evaluations;        including not changing their antiretroviral regimen (unless        medically indicated) during the study period.    -   CD4+ T-cell count >500 cell per millimeter cubed (cells/mm3)    -   CD4+ T-cell nadir of >400 cells/mm3    -   HIV viral load <1,000 copies per milliliter (mL)

Exclusion Criteria:

-   -   Any viral hepatitis    -   Acute HIV infection    -   HIV viral load >1,000 copies/mL    -   Active or recent (prior 6 months) AIDS defining complication    -   Any change in HIV medications within 12 weeks of entering the        study    -   Cancer or malignancy that has not been in remission for at least        5 years with the exception of successfully treated basal cell        carcinoma of the skin    -   Current diagnosis of NYHA grade 3 or 4 congestive heart failure        or uncontrolled angina or arrhythmias    -   History of bleeding problems    -   Use of chronic steroids in past 30 days    -   Pregnant or breast feeding    -   Active drug or alcohol abuse    -   Serious illness in past 30 days    -   Currently participating in another clinical trial or any prior        gene therapy        Safety assessments    -   Acute infusion reaction    -   Post-infusion safety follow-up

Efficacy Assessments—Phase I

-   -   Number and frequency of modified CD4 T cells.    -   Durability of modified CD4 T cells.    -   In vitro response to Gag peptide restimulation (ICS assay) as a        measure of memory T cell function.    -   Polyfunctional anti-HIV CD8 T cell responses compare to pre- and        post-vaccination time points.    -   Frequency of CD4 T cells making doubly spliced HIV mRNA after in        vitro stimulation.

Efficacy Assessments—Phase II

-   -   Number and frequency of genetically modified CD4 T cells.    -   Maintenance of viral suppression (<2,000 vRNA copies per ml but        2 consecutive weekly draws not exceeding 5×10⁴ vRNA copies per        ml are permitted) with Maraviroc monotherapy.    -   Continued virus suppression during and after Maraviroc        withdrawal.    -   Stable CD4 T cell count.

AGT103T Consists of Up to 1×10¹⁰ Genetically Modified, Autologous CD4+ TCells Containing ≥5×10⁷ HIV-Specific CD4 T Cells that are AlsoTransduced with AGT103 Lentivirus Vector.

A Phase I clinical trial will test the safety and feasibility ofinfusing ex vivo modified autologous CD4 T cells (AGT103T) in adultresearch participants with confirmed HIV infection, CD4+ T-cellcounts >600 cells per mm³ of blood and stable virus suppression below200 copies per ml of plasma while on cART. Up to 40 study participantsreceive two doses by intramuscular injection 8 weeks apart, ofrecombinant modified vaccinia Ankara (rMVA) expressing HIV Gag, Pol andEnv proteins. Seven to 10 days after the second immunization a bloodsample is collected for in vitro testing to measure the frequency ofCD4+ T-cells that respond to stimulation with a pool of overlapping,synthetic peptides representing the HIV-1 Gag polyprotein. Subjects inthe upper half of vaccine responders, based on measuring the frequencyof Gag-specific CD4 T cells are enrolled in the gene therapy arm andsubjects in the lower half of responders do not continue in the study.We anticipate that the cut-off for higher responders is a HIV-specificCD4+ T cell frequency ≥0.065% of total CD4 T cells. Subjects enrolledinto the gene therapy arm of our trial undergo leukapheresis and theCD4+ T cells are enriched by negative selection. The enriched CD4 subsetis admixed with 10% the number of cells from the CD4-negative subset toprovide a source and antigen-presenting cells. The enriched CD4 T cellsare stimulated with HIV Gag peptides plus interleukin-2 andinterleukin-12 for 12 days, then stimulated again with beads decoratedwith CD3/CD28 bispecific antibody. The antiretroviral drug Saquinavir isincluded at 100 nM to prevent emergence of autologous HIV during ex vivoculture. One day after CD3/CD28 stimulation cells are transduced withAGT103 at multiplicity of infection between 1 and 10. The transducedcells are cultured for an additional 7-14 days during which time theyexpand by polyclonal proliferation. The culture period is ended byharvesting and washing cells, setting aside aliquots for potency andsafety release assays, and resuspending the remaining cells incryopreservation medium. A single dose is ≤1×10¹⁰ autologous cellsenriched for the CD4+ T cell subset. The potency assay measures thefrequency of CD4 T cells that respond to peptide stimulation byexpressing interferon-gamma. Other release criteria include that theproduct must include ≥0.5×10⁷ HIV-specific CD4 T cells that are alsotransduced with AGT103. Another release criterion is that the number ofAGT103 genome copies per cell must not exceed 3. Five days beforeinfusion with AGT103T subjects receive one dose of busulfuram (orCytoxan) conditioning regimen followed by infusion of ≤1×10¹⁰ enrichedand genetically modified CD4 T cell.

A Phase II study will evaluate efficacy of AGT103T cell therapy. PhaseII study participants include individuals enrolled previously in ourPhase I study who were judged to have successful and stable engraftmentof genetically modified, autologous, HIV-specific CD4 T cells andclinical responses defined as positive changes in parameters monitoredas described in efficacy assessments (1.3.). Study participants will beasked to add Maraviroc to their existing regimen of antiretroviralmedication. Maraviroc is a CCR5 antagonist that will enhance theeffectiveness of genetic therapy directed at reducing CCR5 levels. Oncethe Maraviroc regimen is in place subjects will be asked to discontinuethe previous antiretroviral drug regimen and only maintain Maravirocmonotherapy for 28 days or until plasma viral RNA levels exceed 10,000per ml on 2 sequential weekly blood draws. Persistently high viremiarequires participants to return to their original antiretroviral drugregimen with or without Maraviroc according to recommendations of theirHIV care physician.

If participants remain HIV suppressed (below 2,000 vRNA copies per ml ofplasma) for >28 days on Maraviroc monotherapy, they will be asked togradually reduce Maraviroc dosing over a period of 4 weeks followed byintensive monitoring for an additional 28 days. Subjects who maintainedHIV suppression with Maraviroc monotherapy are considered to have afunctional cure. Subjects who maintain HIV suppression even afterMaraviroc withdrawal also have a functional cure. Monthly monitoring for6 months followed by less intensive monitoring will establish thedurability of functional cure.

Sequences

The following sequences are referred to herein:

SEQ ID NO: Description Sequence 1 miR30 CCR5AGGTATATTGCTGTTGACAGTGAGCGACTGTAAACTGAGCTTGCTCTACTGTGAAGCCACAGATGGGTAGAGCAAGCACAGTTTACCGCTGCCTACTGCCTCGGACTTCAAGGGGCTT 2 miR21 VifCATCTCCATGGCTGTACCACCTTGTCGGGGGATGTGTACTTCTGAACTTGTGTTGAATCTCATGGAGTTCAGAAGAACACATCCGCACTGACATTTTGGTATCTTTCATCTGACCA 3 miR185 TatGGGCCTGGCTCGAGCAGGGGGCGAGGGATTCCGCTTCTTC CTGCCATAGCGTGGTCCCCTCCCCTATGGCAGGCAGAAGCGGCACCTTCCCTCCC AATGACCGCGTCTTCGTCG 4Elongation CCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAG Factor-1 alphaTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGG (EF1-alpha)GAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTT promoterTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACGCCCCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCG TGA 5 CCR5 targetGAGCAAGCTCAGTTTACA sequence 6 Vif target GGGATGTGTACTTCTGAACTT sequence7 Tat target TCCGCTTCTTCCTGCCATAG sequence 8 TAR decoyCTTGCAATGATGTCGTAATTTGCGTCTTACCTCGTTCTCGAC sequenceAGCGACCAGATCTGAGCCTGGGAGCTCTCTGGCTGTCAGTAAGCTGGTACAGAAGGTTGACGAAAATTCTTACTGAGCAAG AAA 9 Rev/Tat targetGCGGAGACAGCGACGAAGAGC sequence 10 Rev/TatGCGGAGACAGCGACGAAGAGCTTCAAGAGAGCTCTTCGTC shRNA GCTGTCTCCGCTTTTT sequence11 Gag target GAAGAAATGATGACAGCAT sequence 12 Gag shRNAGAAGAAATGATGACAGCATTTCAAGAGAATGCTGTCATCA sequence TTTCTTCTTTTT 13Pol target CAGGAGCAGATGATACAG sequence 14 Pol shRNACAGGAGATGATACAGTTCAAGAGACTGTATCATCTGCTCCT sequence GTTTTT 15 CCR5 targetGTGTCAAGTCCAATCTATG sequence #1 16 CCR5 shRNAGTGTCAAGTCCAATCTATGTTCAAGAGACATAGATTGGACT sequence #1 TGACACTTTTT 17CCR5 target GAGCATGACTGACATCTAC sequence #2 18 CCR5 shRNAGAGCATGACTGACATCTACTTCAAGAGAGTAGATGTCAGT sequence #2 CATGCTCTTTTT 19CCR5 target GTAGCTCTAACAGGTTGGA sequence #3 20 CCR5 shRNAGTAGCTCTAACAGGTTGGATTCAAGAGATCCAACCTGTTAG sequence #3 AGCTACTTTTT 21CCR5 target GTTCAGAAACTACCTCTTA sequence #4 22 CCR5 shRNAGTTCAGAAACTACCTCTTATTCAAGAGATAAGAGGTAGTTT sequence #4 CTGAACTTTTT 23CCR5 target GAGCAAGCTCAGTTTACACC sequence #5 24 CCR5 shRNAGAGCAAGCTCAGTTTACACCTTCAAGAGAGGTGTAAACTG sequence #5 AGCTTGCTCTTTTT 25Homo sapiens ATGGATTATCAAGTGTCAAGTCCAATCTATGACATCAATTA CCR5 gene,TTATACATCGGAGCCCTGCCAAAAAATCAATGTGAAGCAA sequence 1ATCGCAGCCCGCCTCCTGCCTCCGCTCTACTCACTGGTGTT CATCTTTGGTTTTGTGGGC 26Homo sapiens AACATGCTGGTCATCCTCATCCTGATAAACTGCAAAAGGCT CCR5 gene,GAAGAGCATGACTGACATCTACCTGCTCAACCTGGCCATCT sequence 2CTGACCTGTTTTTCCTTCTTACTGTCCCCTTCTGGGCTCACTATGCTGCCGCCCAGTGGGACTTTGGAAATACAATGTGTCAACTCTTGACAGGGCTCTATTTTATAGGCTTCTTCTCTGGAATCTTCTTCATCATCCTCCTGACAATCGATAGGTACCTGGCTGTCGTCCATGCTGTGTTTGCTTTAAAAGCCAGGACGGTCACCTTTGGGGTGGTGACAAGTGTGATCACTTGGGTGGTGGCTGTGTTTGCGTCTCTCCCAGGAATCATCTTTACCAGATCTCAAAAAGAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCATACAGTCAGTATCAATTCTGGAAGAATTTCCAGACATTAAAGATAGTCATCTTGGGGCTGGTCCTGCCGCTGCTTGTCATGGTCATCTGCTACTCGGGAATCCTAAAAACTCTGCTTCGGTGTCGAAATGAGAAGAAGAGGCACAGGGCTGTGAGGCTTATCTTCACCATCATGATTGTTTATTTTCTCTTCTGGGCTCCCTACAACAT TGTCCTTCTCCTGAAC 27Homo sapiens ACCTTCCAGGAATTCTTTGGCCTGAATAATTGCAGTAGCTC CCR5 gene,TAACAGGTTGGACCAAGCTATGCAGGTGA sequence 3 28 Homo sapiensCAGAGACTCTTGGGATGACGCACTGCTGCATCAACCCCATC CCR5 gene,ATCTATGCCTTTGTCGGGGAGAAGTTCAGAAACTACCTCTT sequence 4AGTCTTCTTCCAAAAGCACATTGCCAAACGCTTCTGCAAAT GCTGTTCTATTTTCCAG 29Homo sapiens CAAGAGGCTCCCGAGCGAGCAAGCTCAGTTTACACCCGAT CCR5 gene,CCACTGGGGAGCAGGAAATATCTGTGGGCTTGTGA sequence 5 30 CD4 promoterTGTTGGGGTTCAAATTTGAGCCCCAGCTGTTAGCCCTCTGC sequenceAAAGAAAAAAAAAAAAAAAAAAGAACAAAGGGCCTAGATTTCCCTTCTGAGCCCCACCCTAAGATGAAGCCTCTTCTTTCAAGGGAGTGGGGTTGGGGTGGAGGCGGATCCTGTCAGCTTTGCTCTCTCTGTGGCTGGCAGTTTCTCCAAAGGGTAACAGGTGTCAGCTGGCTGAGCCTAGGCTGAACCCTGAGACATGCTACCTCTGTCTTCTCATGGCTGGAGGCAGCCTTTGTAAGTCACAGAAAGTAGCTGAGGGGCTCTGGAAAAAAGACAGCCAGGGTGGAGGTAGATTGGTCTTTGACTCCTGATTTAAGCCTGATTCTGCTTAACTTTTTCCCTTGACTTTGGCATTTTCACTTTGACATGTTCCCTGAGAGCCTGGGGGGTGGGGAACCCAGCTCCAGCTGGTGACGTTTGGGGCCGGCCCAGGCCTAGGGTGTGGAGGAGCCTTGCCATCGGGCTTCCTGTCTCTCTTCATTTAAG CACGACTCTGCAGA 31 miR30-AGGTATATTGCTGTTGACAGTGAGCGACTGTAAACTGAGCT CCR5/miR21-TGCTCTACTGTGAAGCCACAGATGGGTAGAGCAAGCACAG Vif/miR185TTTACCGCTGCCTACTGCCTCGGACTTCAAGGGGCTTCCCG TatGGCATCTCCATGGCTGTACCACCTTGTCGGGGGATGTGTAC microRNATTCTGAACTTGTGTTGAATCTCATGGAGTTCAGAAGAACAC clusterATCCGCACTGACATTTTGGTATCTTTCATCTGACCAGCTAG sequenceCGGGCCTGGCTCGAGCAGGGGGCGAGGGATTCCGCTTCTTCCTGCCATAGCGTGGTCCCCTCCCCTATGGCAGGCAGAAGCGGCACCTTCCCTCCCAATGACCGCGTCTTCGTC 32 Long WPREAATCAACCTCTGATTACAAAATTTGTGAAAGATTGACTGGT sequenceATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGC CGCCTCCCCGCCT 33 ElongationCCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAG Factor-1 alphaTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGG (EF1-alpha)GAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTT promoter;TTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGT miR30CCR5;GTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCC miR21Vif;CTTGCGTGCCTTGAATTACTTCCACGCCCCTGGCTGCAGTA miR185TatCGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCG TGATGTACAAGGTATATTGCTGTTGACAGTGAGCGACTGTAAACTGAGCTTGCTCTACTGTGAAGCCACAGATGGGTAGAGCAAGCACAGTTTACCGCTGCCTACTGCCTCGGACTTCAAGGGGCTTCCCGGGCATCTCCATGGCTGTACCACCTTGTCGGGGGATGTGTACTTCTGAACTTGTGTTGAATCTCATGGAGTTCAGAAGAACACATCCGCACTGACATTTTGGTATCTTTCATCTGACCAGCTAGCGGGCCTGGCTCGAGCAGGGGGCGAGGGATTCCGCTTCTTCCTGCCATAGCGTGGTCCCCTCCCCTATGGCAGGCAGAAGCGGCACCTTCCCTCCCAATGACCGCGTCTTCGTC 34 Rous SarcomaGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAA virus (RSV)CGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCA promoterCCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTT AAGTGCCTAGCTCGATACAATAAACG 355′ Long GGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCT terminalGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTT repeat (LTR)GCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTG TGGAAAATCTCTAGCA 36Psi Packaging TACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGA signal G 37Rev response AGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCA elementCTATGGGCGCAGCCTCAATGACGCTGACGGTACAGGCCAG (RRE)ACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCT GTGGAAAGATACCTAAAGGATCAACAGCTCC38 Central TTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGG polypurineGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTA tract (cPPT)AAGAATTACAAAAACAAATTACAAAATTCAAAATTTTA 39 3′ delta LTRTGGAAGGGCTAATTCACTCCCAACGAAGATAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCAT GTCA 40 Helper/Rev;TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATA CMV earlyGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAAT (CAG)GGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGAC enhancer;GTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGG EnhanceACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAAC TranscriptionTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTG GCAGTACATCTACGTATTAGTCATC 41Helper/Rev; GCTATTACCATGGGTCGAGGTGAGCCCCACGTTCTGCTTCA Chicken betaCTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATT actin (CAG)TATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGG promoter;GGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGA TranscriptionGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCG GCGGGCG 42 Helper/Rev;GGAGTCGCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCGCGC Chicken betaCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTAC actin intron;TCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGG Enhance geneCTGTAATTAGCGCTTGGTTTAATGACGGCTCGTTTCTTTTCT expressionGTGGCTGCGTGAAAGCCTTAAAGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGGCGGTCGGGCTGTAACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTGCGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCATCTCCAGCCTCGGGGCTGCCGCAGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGG 43 Helper/Rev;ATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAG HIV Gag;ATCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAA Viral capsidAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGAAAAAAGCACAGCAAGCAGCAGCTGACACAGGACACAGCAATCAGGTCAGCCAAAATTACCCTATAGTGCAGAACATCCAGGGGCAAATGGTACATCAGGCCATATCACCTAGAACTTTAAATGCATGGGTAAAAGTAGTAGAAGAGAAGGCTTTCAGCCCAGAAGTGATACCCATGTTTTCAGCATTATCAGAAGGAGCCACCCCACAAGATTTAAACACCATGCTAAACACAGTGGGGGGACATCAAGCAGCCATGCAAATGTTAAAAGAGACCATCAATGAGGAAGCTGCAGAATGGGATAGAGTGCATCCAGTGCATGCAGGGCCTATTGCACCAGGCCAGATGAGAGAACCAAGGGGAAGTGACATAGCAGGAACTACTAGTACCCTTCAGGAACAAATAGGATGGATGACACATAATCCACCTATCCCAGTAGGAGAAATCTATAAAAGATGGATAATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTACCAGCATTCTGGACATAAGACAAGGACCAAAGGAACCCTTTAGAGACTATGTAGACCGATTCTATAAAACTCTAAGAGCCGAGCAAGCTTCACAAGAGGTAAAAAATTGGATGACAGAAACCTTGTTGGTCCAAAATGCGAACCCAGATTGTAAGACTATTTTAAAAGCATTGGGACCAGGAGCGACACTAGAAGAAATGATGACAGCATGTCAGGGAGTGGGGGGACCCGGCCATAAAGCAAGAGTTTTGGCTGAAGCAATGAGCCAAGTAACAAATCCAGCTACCATAATGATACAGAAAGGCAATTTTAGGAACCAAAGAAAGACTGTTAAGTGTTTCAATTGTGGCAAAGAAGGGCACATAGCCAAAAATTGCAGGGCCCCTAGGAAAAAGGGCTGTTGGAAATGTGGAAAGGAAGGACACCAAATGAAAGATTGTACTGAGAGACAGGCTAATTTTTTAGGGAAGATCTGGCCTTCCCACAAGGGAAGGCCAGGGAATTTTCTTCAGAGCAGACCAGAGCCAACAGCCCCACCAGAAGAGAGCTTCAGGTTTGGGGAAGAGACAACAACTCCCTCTCAGAAGCAGGAGCCGATAGACAAGGAACTGTATCCTTTAGCTTCCCTCAGATCACTCTTTGGCAG CGACCCCTCGTCACAATAA 44Helper/Rev; ATGAATTTGCCAGGAAGATGGAAACCAAAAATGATAGGGG HIV Pol;GAATTGGAGGTTTTATCAAAGTAGGACAGTATGATCAGAT Protease andACTCATAGAAATCTGCGGACATAAAGCTATAGGTACAGTA reverseTTAGTAGGACCTACACCTGTCAACATAATTGGAAGAAATCT transcriptaseGTTGACTCAGATTGGCTGCACTTTAAATTTTCCCATTAGTCCTATTGAGACTGTACCAGTAAAATTAAAGCCAGGAATGGATGGCCCAAAAGTTAAACAATGGCCATTGACAGAAGAAAAAATAAAAGCATTAGTAGAAATTTGTACAGAAATGGAAAAGGAAGGAAAAATTTCAAAAATTGGGCCTGAAAATCCATACAATACTCCAGTATTTGCCATAAAGAAAAAAGACAGTACTAAATGGAGAAAATTAGTAGATTTCAGAGAACTTAATAAGAGAACTCAAGATTTCTGGGAAGTTCAATTAGGAATACCACATCCTGCAGGGTTAAAACAGAAAAAATCAGTAACAGTACTGGATGTGGGCGATGCATATTTTTCAGTTCCCTTAGATAAAGACTTCAGGAAGTATACTGCATTTACCATACCTAGTATAAACAATGAGACACCAGGGATTAGATATCAGTACAATGTGCTTCCACAGGGATGGAAAGGATCACCAGCAATATTCCAGTGTAGCATGACAAAAATCTTAGAGCCTTTTAGAAAACAAAATCCAGACATAGTCATCTATCAATACATGGATGATTTGTATGTAGGATCTGACTTAGAAATAGGGCAGCATAGAACAAAAATAGAGGAACTGAGACAACATCTGTTGAGGTGGGGATTTACCACACCAGACAAAAAACATCAGAAAGAACCTCCATTCCTTTGGATGGGTTATGAACTCCATCCTGATAAATGGACAGTACAGCCTATAGTGCTGCCAGAAAAGGACAGCTGGACTGTCAATGACATACAGAAATTAGTGGGAAAATTGAATTGGGCAAGTCAGATTTATGCAGGGATTAAAGTAAGGCAATTATGTAAACTTCTTAGGGGAACCAAAGCACTAACAGAAGTAGTACCACTAACAGAAGAAGCAGAGCTAGAACTGGCAGAAAACAGGGAGATTCTAAAAGAACCGGTACATGGAGTGTATTATGACCCATCAAAAGACTTAATAGCAGAAATACAGAAGCAGGGGCAAGGCCAATGGACATATCAAATTTATCAAGAGCCATTTAAAAATCTGAAAACAGGAAAATATGCAAGAATGAAGGGTGCCCACACTAATGATGTGAAACAATTAACAGAGGCAGTACAAAAAATAGCCACAGAAAGCATAGTAATATGGGGAAAGACTCCTAAATTTAAATTACCCATACAAAAGGAAACATGGGAAGCATGGTGGACAGAGTATTGGCAAGCCACCTGGATTCCTGAGTGGGAGTTTGTCAATACCCCTCCCTTAGTGAAGTTATGGTACCAGTTAGAGAAAGAACCCATAATAGGAGCAGAAACTTTCTATGTAGATGGGGCAGCCAATAGGGAAACTAAATTAGGAAAAGCAGGATATGTAACTGACAGAGGAAGACAAAAAGTTGTCCCCCTAACGGACACAACAAATCAGAAGACTGAGTTACAAGCAATTCATCTAGCTTTGCAGGATTCGGGATTAGAAGTAAACATAGTGACAGACTCACAATATGCATTGGGAATCATTCAAGCACAACCAGATAAGAGTGAATCAGAGTTAGTCAGTCAAATAATAGAGCAGTTAATAAAAAAGGAAAAAGTCTACCTGGCATGGGTACCAGCACACAAAGGAATTGGAGGAAATGAACAAGTAGATGGGTTGGTCAGTGC TGGAATCAGGAAAGTACTA 45Helper Rev; TTTTTAGATGGAATAGATAAGGCCCAAGAAGAACATGAGA HIVAATATCACAGTAATTGGAGAGCAATGGCTAGTGATTTTAAC Integrase;CTACCACCTGTAGTAGCAAAAGAAATAGTAGCCAGCTGTG Integration ofATAAATGTCAGCTAAAAGGGGAAGCCATGCATGGACAAGT viral RNAAGACTGTAGCCCAGGAATATGGCAGCTAGATTGTACACATTTAGAAGGAAAAGTTATCTTGGTAGCAGTTCATGTAGCCAGTGGATATATAGAAGCAGAAGTAATTCCAGCAGAGACAGGGCAAGAAACAGCATACTTCCTCTTAAAATTAGCAGGAAGATGGCCAGTAAAAACAGTACATACAGACAATGGCAGCAATTTCACCAGTACTACAGTTAAGGCCGCCTGTTGGTGGGCGGGGATCAAGCAGGAATTTGGCATTCCCTACAATCCCCAAAGTCAAGGAGTAATAGAATCTATGAATAAAGAATTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTTAAGACAGCAGTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTTGGAAAGGACCAGCAAAGCTCCTCTGGAAAGGTGAAGGGGCAGTAGTAATACAAGATAATAGTGACATAAAAGTAGTGCCAAGAAGAAAAGCAAAGATCATCAGGGATTATGGAAAACAGATGGCAGGTGATGATTGTGTG GCAAGTAGACAGGATGAGGATTAA 46Helper/Rev; AGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCA HIV RRE;CTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAG Binds RevACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGC elementTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCT 47 Helper/Rev;ATGGCAGGAAGAAGCGGAGACAGCGACGAAGAACTCCTC HIV Rev;AAGGCAGTCAGACTCATCAAGTTTCTCTATCAAAGCAACCC NuclearACCTCCCAATCCCGAGGGGACCCGACAGGCCCGAAGGAAT export andAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCAT stabilize viralTCGATTAGTGAACGGATCCTTAGCACTTATCTGGGACGATC mRNATGCGGAGCCTGTGCCTCTTCAGCTACCACCGCTTGAGAGACTTACTCTTGATTGTAACGAGGATTGTGGAACTTCTGGGACGCAGGGGGTGGGAAGCCCTCAAATATTGGTGGAATCTCCTA CAATATTGGAGTCAGGAGCTAAAGAATAG48 Helper/Rev; AGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGA Rabbit betaAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTT globin poly A;ATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCA RNA stabilityCTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCATATGCTGGCTGCCATGAACAAAGGTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGAAGATC 49 Helper; CMVTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATA early (CAG)GCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAAT enhancer;GGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGAC EnhanceGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGG transcriptionACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTG GCAGTACATCTACGTATTAGTCATC 50Helper; GCTATTACCATGGGTCGAGGTGAGCCCCACGTTCTGCTTCA Chicken betaCTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATT actin (CAG)TATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGG promoter;GGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGA TranscriptionGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCG GCGGGCG 51 Helper;GGAGTCGCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCGCGC Chicken betaCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTAC actin intron;TCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGG Enhance geneCTGTAATTAGCGCTTGGTTTAATGACGGCTCGTTTCTTTTCT expressionGTGGCTGCGTGAAAGCCTTAAAGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGGCGGTCGGGCTGTAACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTGCGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCATCTCCAGCCTCGGGGCTGCCGCAGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGG 52 Helper; HIVATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAG Gag; ViralATCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAA capsidAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGAAAAAAGCACAGCAAGCAGCAGCTGACACAGGACACAGCAATCAGGTCAGCCAAAATTACCCTATAGTGCAGAACATCCAGGGGCAAATGGTACATCAGGCCATATCACCTAGAACTTTAAATGCATGGGTAAAAGTAGTAGAAGAGAAGGCTTTCAGCCCAGAAGTGATACCCATGTTTTCAGCATTATCAGAAGGAGCCACCCCACAAGATTTAAACACCATGCTAAACACAGTGGGGGGACATCAAGCAGCCATGCAAATGTTAAAAGAGACCATCAATGAGGAAGCTGCAGAATGGGATAGAGTGCATCCAGTGCATGCAGGGCCTATTGCACCAGGCCAGATGAGAGAACCAAGGGGAAGTGACATAGCAGGAACTACTAGTACCCTTCAGGAACAAATAGGATGGATGACACATAATCCACCTATCCCAGTAGGAGAAATCTATAAAAGATGGATAATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTACCAGCATTCTGGACATAAGACAAGGACCAAAGGAACCCTTTAGAGACTATGTAGACCGATTCTATAAAACTCTAAGAGCCGAGCAAGCTTCACAAGAGGTAAAAAATTGGATGACAGAAACCTTGTTGGTCCAAAATGCGAACCCAGATTGTAAGACTATTTTAAAAGCATTGGGACCAGGAGCGACACTAGAAGAAATGATGACAGCATGTCAGGGAGTGGGGGGACCCGGCCATAAAGCAAGAGTTTTGGCTGAAGCAATGAGCCAAGTAACAAATCCAGCTACCATAATGATACAGAAAGGCAATTTTAGGAACCAAAGAAAGACTGTTAAGTGTTTCAATTGTGGCAAAGAAGGGCACATAGCCAAAAATTGCAGGGCCCCTAGGAAAAAGGGCTGTTGGAAATGTGGAAAGGAAGGACACCAAATGAAAGATTGTACTGAGAGACAGGCTAATTTTTTAGGGAAGATCTGGCCTTCCCACAAGGGAAGGCCAGGGAATTTTCTTCAGAGCAGACCAGAGCCAACAGCCCCACCAGAAGAGAGCTTCAGGTTTGGGGAAGAGACAACAACTCCCTCTCAGAAGCAGGAGCCGATAGACAAGGAACTGTATCCTTTAGCTTCCCTCAGATCACTCTTTGGCAG CGACCCCTCGTCACAATAA 53Helper; HIV ATGAATTTGCCAGGAAGATGGAAACCAAAAATGATAGGGG Pol; ProteaseGAATTGGAGGTTTTATCAAAGTAGGACAGTATGATCAGAT and reverseACTCATAGAAATCTGCGGACATAAAGCTATAGGTACAGTA transcriptaseTTAGTAGGACCTACACCTGTCAACATAATTGGAAGAAATCTGTTGACTCAGATTGGCTGCACTTTAAATTTTCCCATTAGTCCTATTGAGACTGTACCAGTAAAATTAAAGCCAGGAATGGATGGCCCAAAAGTTAAACAATGGCCATTGACAGAAGAAAAAATAAAAGCATTAGTAGAAATTTGTACAGAAATGGAAAAGGAAGGAAAAATTTCAAAAATTGGGCCTGAAAATCCATACAATACTCCAGTATTTGCCATAAAGAAAAAAGACAGTACTAAATGGAGAAAATTAGTAGATTTCAGAGAACTTAATAAGAGAACTCAAGATTTCTGGGAAGTTCAATTAGGAATACCACATCCTGCAGGGTTAAAACAGAAAAAATCAGTAACAGTACTGGATGTGGGCGATGCATATTTTTCAGTTCCCTTAGATAAAGACTTCAGGAAGTATACTGCATTTACCATACCTAGTATAAACAATGAGACACCAGGGATTAGATATCAGTACAATGTGCTTCCACAGGGATGGAAAGGATCACCAGCAATATTCCAGTGTAGCATGACAAAAATCTTAGAGCCTTTTAGAAAACAAAATCCAGACATAGTCATCTATCAATACATGGATGATTTGTATGTAGGATCTGACTTAGAAATAGGGCAGCATAGAACAAAAATAGAGGAACTGAGACAACATCTGTTGAGGTGGGGATTTACCACACCAGACAAAAAACATCAGAAAGAACCTCCATTCCTTTGGATGGGTTATGAACTCCATCCTGATAAATGGACAGTACAGCCTATAGTGCTGCCAGAAAAGGACAGCTGGACTGTCAATGACATACAGAAATTAGTGGGAAAATTGAATTGGGCAAGTCAGATTTATGCAGGGATTAAAGTAAGGCAATTATGTAAACTTCTTAGGGGAACCAAAGCACTAACAGAAGTAGTACCACTAACAGAAGAAGCAGAGCTAGAACTGGCAGAAAACAGGGAGATTCTAAAAGAACCGGTACATGGAGTGTATTATGACCCATCAAAAGACTTAATAGCAGAAATACAGAAGCAGGGGCAAGGCCAATGGACATATCAAATTTATCAAGAGCCATTTAAAAATCTGAAAACAGGAAAATATGCAAGAATGAAGGGTGCCCACACTAATGATGTGAAACAATTAACAGAGGCAGTACAAAAAATAGCCACAGAAAGCATAGTAATATGGGGAAAGACTCCTAAATTTAAATTACCCATACAAAAGGAAACATGGGAAGCATGGTGGACAGAGTATTGGCAAGCCACCTGGATTCCTGAGTGGGAGTTTGTCAATACCCCTCCCTTAGTGAAGTTATGGTACCAGTTAGAGAAAGAACCCATAATAGGAGCAGAAACTTTCTATGTAGATGGGGCAGCCAATAGGGAAACTAAATTAGGAAAAGCAGGATATGTAACTGACAGAGGAAGACAAAAAGTTGTCCCCCTAACGGACACAACAAATCAGAAGACTGAGTTACAAGCAATTCATCTAGCTTTGCAGGATTCGGGATTAGAAGTAAACATAGTGACAGACTCACAATATGCATTGGGAATCATTCAAGCACAACCAGATAAGAGTGAATCAGAGTTAGTCAGTCAAATAATAGAGCAGTTAATAAAAAAGGAAAAAGTCTACCTGGCATGGGTACCAGCACACAAAGGAATTGGAGGAAATGAACAAGTAGATGGGTTGGTCAGTGC TGGAATCAGGAAAGTACTA 54Helper; HIV TTTTTAGATGGAATAGATAAGGCCCAAGAAGAACATGAGA Integrase;AATATCACAGTAATTGGAGAGCAATGGCTAGTGATTTTAAC Integration ofCTACCACCTGTAGTAGCAAAAGAAATAGTAGCCAGCTGTG viral RNAATAAATGTCAGCTAAAAGGGGAAGCCATGCATGGACAAGTAGACTGTAGCCCAGGAATATGGCAGCTAGATTGTACACATTTAGAAGGAAAAGTTATCTTGGTAGCAGTTCATGTAGCCAGTGGATATATAGAAGCAGAAGTAATTCCAGCAGAGACAGGGCAAGAAACAGCATACTTCCTCTTAAAATTAGCAGGAAGATGGCCAGTAAAAACAGTACATACAGACAATGGCAGCAATTTCACCAGTACTACAGTTAAGGCCGCCTGTTGGTGGGCGGGGATCAAGCAGGAATTTGGCATTCCCTACAATCCCCAAAGTCAAGGAGTAATAGAATCTATGAATAAAGAATTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTTAAGACAGCAGTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTTGGAAAGGACCAGCAAAGCTCCTCTGGAAAGGTGAAGGGGCAGTAGTAATACAAGATAATAGTGACATAAAAGTAGTGCCAAGAAGAAAAGCAAAGATCATCAGGGATTATGGAAAACAGATGGCAGGTGATGATTGTGTG GCAAGTAGACAGGATGAGGATTAA 55Helper; HIV AGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCA RRE; BindsCTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAG Rev elementACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCT 56 Helper;AGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGA Rabbit betaAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTT globin poly A;ATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCA RNA stabilityCTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCATATGCTGGCTGCCATGAACAAAGGTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGAAGATC 57 Rev; RSVATGGCAGGAAGAAGCGGAGACAGCGACGAAGAACTCCTC promoter;AAGGCAGTCAGACTCATCAAGTTTCTCTATCAAAGCAACCC TranscriptionACCTCCCAATCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCCTTAGCACTTATCTGGGACGATCTGCGGAGCCTGTGCCTCTTCAGCTACCACCGCTTGAGAGACTTACTCTTGATTGTAACGAGGATTGTGGAACTTCTGGGACGCAGGGGGTGGGAAGCCCTCAAATATTGGTGGAATCTCCTA CAATATTGGAGTCAGGAGCTAAAGAATAG58 Rev; HIV ATGGCAGGAAGAAGCGGAGACAGCGACGAAGAACTCCTC Rev; NuclearAAGGCAGTCAGACTCATCAAGTTTCTCTATCAAAGCAACCC export andACCTCCCAATCCCGAGGGGACCCGACAGGCCCGAAGGAAT stabilize viralAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCAT mRNATCGATTAGTGAACGGATCCTTAGCACTTATCTGGGACGATCTGCGGAGCCTGTGCCTCTTCAGCTACCACCGCTTGAGAGACTTACTCTTGATTGTAACGAGGATTGTGGAACTTCTGGGACGCAGGGGGTGGGAAGCCCTCAAATATTGGTGGAATCTCCTA CAATATTGGAGTCAGGAGCTAAAGAATAG59 Rev; Rabbit AGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGA beta globinAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTT poly A; RNAATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCA stabilityCTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATC 60 Envelope;ACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGG CMVGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACA promoter;TAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACG TranscriptionACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTAT ATAAGC 61 Envelope;GTGAGTTTGGGGACCCTTGATTGTTCTTTCTTTTTCGCTATT Beta globinGTAAAATTCATGTTATATGGAGGGGGCAAAGTTTTCAGGGT intron;GTTGTTTAGAATGGGAAGATGTCCCTTGTATCACCATGGAC Enhance geneCCTCATGATAATTTTGTTTCTTTCACTTTCTACTCTGTTGAC expressionAACCATTGTCTCCTCTTATTTTCTTTTCATTTTCTGTAACTTTTTCGTTAAACTTTAGCTTGCATTTGTAACGAATTTTTAAATTCACTTTTGTTTATTTGTCAGATTGTAAGTACTTTCTCTAATCACTTTTTTTTCAAGGCAATCAGGGTATATTATATTGTACTTCAGCACAGTTTTAGAGAACAATTGTTATAATTAAATGATAAGGTAGAATATTTCTGCATATAAATTCTGGCTGGCGTGGAAATATTCTTATTGGTAGAAACAACTACACCCTGGTCATCATCCTGCCTTTCTCTTTATGGTTACAATGATATACACTGTTTGAGATGAGGATAAAATACTCTGAGTCCAAACCGGGCCCCTCTGCTAACCATGTTCATGCCTTCTTCTCTTTCCTACAG 62 Envelope;ATGAAGTGCCTTTTGTACTTAGCCTTTTTATTCATTGGGGTG VSV-G;AATTGCAAGTTCACCATAGTTTTTCCACACAACCAAAAAGG GlycoproteinAAACTGGAAAAATGTTCCTTCTAATTACCATTATTGCCCGT envelope-cellCAAGCTCAGATTTAAATTGGCATAATGACTTAATAGGCACA entryGCCTTACAAGTCAAAATGCCCAAGAGTCACAAGGCTATTCAAGCAGACGGTTGGATGTGTCATGCTTCCAAATGGGTCACTACTTGTGATTTCCGCTGGTATGGACCGAAGTATATAACACATTCCATCCGATCCTTCACTCCATCTGTAGAACAATGCAAGGAAAGCATTGAACAAACGAAACAAGGAACTTGGCTGAATCCAGGCTTCCCTCCTCAAAGTTGTGGATATGCAACTGTGACGGATGCCGAAGCAGTGATTGTCCAGGTGACTCCTCACCATGTGCTGGTTGATGAATACACAGGAGAATGGGTTGATTCACAGTTCATCAACGGAAAATGCAGCAATTACATATGCCCCACTGTCCATAACTCTACAACCTGGCATTCTGACTATAAGGTCAAAGGGCTATGTGATTCTAACCTCATTTCCATGGACATCACCTTCTTCTCAGAGGACGGAGAGCTATCATCCCTGGGAAAGGAGGGCACAGGGTTCAGAAGTAACTACTTTGCTTATGAAACTGGAGGCAAGGCCTGCAAAATGCAATACTGCAAGCATTGGGGAGTCAGACTCCCATCAGGTGTCTGGTTCGAGATGGCTGATAAGGATCTCTTTGCTGCAGCCAGATTCCCTGAATGCCCAGAAGGGTCAAGTATCTCTGCTCCATCTCAGACCTCAGTGGATGTAAGTCTAATTCAGGACGTTGAGAGGATCTTGGATTATTCCCTCTGCCAAGAAACCTGGAGCAAAATCAGAGCGGGTCTTCCAATCTCTCCAGTGGATCTCAGCTATCTTGCTCCTAAAAACCCAGGAACCGGTCCTGCTTTCACCATAATCAATGGTACCCTAAAATACTTTGAGACCAGATACATCAGAGTCGATATTGCTGCTCCAATCCTCTCAAGAATGGTCGGAATGATCAGTGGAACTACCACAGAAAGGGAACTGTGGGATGACTGGGCACCATATGAAGACGTGGAAATTGGACCCAATGGAGTTCTGAGGACCAGTTCAGGATATAAGTTTCCTTTATACATGATTGGACATGGTATGTTGGACTCCGATCTTCATCTTAGCTCAAAGGCTCAGGTGTTCGAACATCCTCACATTCAAGACGCTGCTTCGCAACTTCCTGATGATGAGAGTTTATTTTTTGGTGATACTGGGCTATCCAAAAATCCAATCGAGCTTGTAGAAGGTTGGTTCAGTAGTTGGAAAAGCTCTATTGCCTCTTTTTTCTTTATCATAGGGTTAATCATTGGACTATTCTTGGTTCTCCGAGTTGGTATCCATCTTTGCATTAAATTAAAGCACACCAAGAAAAGACAGATTTATACAGACAT AGAGATGA 63 Envelope;AGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGA Rabbit betaAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTT globin poly A;ATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCA RNA stabilityCTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATC 64 Promoter; EF-CCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAG 1TGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACGCCCCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCG TGA 65 Promoter;GGGGTTGGGGTTGCGCCTTTTCCAAGGCAGCCCTGGGTTTG PGKCGCAGGGACGCGGCTGCTCTGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCTGGGTCTCGCACATTCTTCACGTCCGTTCGCAGCGTCACCCGGATCTTCGCCGCTACCCTTGTGGGCCCCCCGGCGACGCTTCCTGCTCCGCCCCTAAGTCGGGAAGGTTCCTTGCGGTTCGCGGCGTGCCGGACGTGACAAACGGAAGCCGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCCAGGGAGCAATGGCAGCGCGCCGACCGCGATGGGCTGTGGCCAATAGCGGCTGCTCAGCAGGGCGCGCCGAGAGCAGCGGCCGGGAAGGGGCGGTGCGGGAGGCGGGGTGTGGGGCGGTAGTGTGGGCCCTGTTCCTGCCCGCGCGGTGTTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCGGCAGTCGGCTCCCTCGTTG ACCGAATCACCGACCTCTCTCCCCAG 66Promoter; GCGCCGGGTTTTGGCGCCTCCCGCGGGCGCCCCCCTCCTCA UbCCGGCGAGCGCTGCCACGTCAGACGAAGGGCGCAGGAGCGTTCCTGATCCTTCCGCCCGGACGCTCAGGACAGCGGCCCGCTGCTCATAAGACTCGGCCTTAGAACCCCAGTATCAGCAGAAGGACATTTTAGGACGGGACTTGGGTGACTCTAGGGCACTGGTTTTCTTTCCAGAGAGCGGAACAGGCGAGGAAAAGTAGTCCCTTCTCGGCGATTCTGCGGAGGGATCTCCGTGGGGCGGTGAACGCCGATGATTATATAAGGACGCGCCGGGTGTGGCACAGCTAGTTCCGTCGCAGCCGGGATTTGGGTCGCGGTTCTTGTTTGTGGATCGCTGTGATCGTCACTTGGTGAGTTGCGGGCTGCTGGGCTGGCCGGGGCTTTCGTGGCCGCCGGGCCGCTCGGTGGGACGGAAGCGTGTGGAGAGACCGCCAAGGGCTGTAGTCTGGGTCCGCGAGCAAGGTTGCCCTGAACTGGGGGTTGGGGGGAGCGCACAAAATGGCGGCTGTTCCCGAGTCTTGAATGGAAGACGCTTGTAAGGCGGGCTGTGAGGTCGTTGAAACAAGGTGGGGGGCATGGTGGGCGGCAAGAACCCAAGGTCTTGAGGCCTTCGCTAATGCGGGAAAGCTCTTATTCGGGTGAGATGGGCTGGGGCACCATCTGGGGACCCTGACGTGAAGTTTGTCACTGACTGGAGAACTCGGGTTTGTCGTCTGGTTGCGGGGGCGGCAGTTATGCGGTGCCGTTGGGCAGTGCACCCGTACCTTTGGGAGCGCGCGCCTCGTCGTGTCGTGACGTCACCCGTTCTGTTGGCTTATAATGCAGGGTGGGGCCACCTGCCGGTAGGTGTGCGGTAGGCTTTTCTCCGTCGCAGGACGCAGGGTTCGGGCCTAGGGTAGGCTCTCCTGAATCGACAGGCGCCGGACCTCTGGTGAGGGGAGGGATAAGTGAGGCGTCAGTTTCTTTGGTCGGTTTTATGTACCTATCTTCTTAAGTAGCTGAAGCTCCGGTTTTGAACTATGCGCTCGGGGTTGGCGAGTGTGTTTTGTGAAGTTTTTTAGGCACCTTTTGAAATGTAATCATTTGGGTCAATAT GTAATTTTCAGTGTTAGACTAGTAAA 67Poly A; SV40 GTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCA 68 Poly A; bGHGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATG CTGGGGATGCGGTGGGCTCTATGG 69HIV Gag; Bal ATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATAGGTGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAGATTAAAACATATAGTATGGGCAAGCAGGGAACTAGAAAGATTCGCAGTCAATCCTGGCCTGTTAGAAACATCAGAAGGCTGCAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTACATCAAAAGATAGAGGTAAAAGACACCAAGGAAGCTTTAGACAAAATAGAGGAAGAGCAAAACAAATGTAAGAAAAAGGCACAGCAAGCAGCAGCTGACACAGGAAACAGCGGTCAGGTCAGCCAAAATTTCCCTATAGTGCAGAACCTCCAGGGGCAAATGGTACATCAGGCCATATCACCTAGAACTTTAAATGCATGGGTAAAAGTAATAGAAGAGAAAGCTTTCAGCCCAGAAGTAATACCCATGTTTTCAGCATTATCAGAAGGAGCCACCCCACAAGATTTAAACACCATGCTAAACACAGTGGGGGGACATCAAGCAGCCATGCAAATGTTAAAAGAACCCATCAATGAGGAAGCTGCAAGATGGGATAGATTGCATCCCGTGCAGGCAGGGCCTGTTGCACCAGGCCAGATAAGAGATCCAAGGGGAAGTGACATAGCAGGAACTACCAGTACCCTTCAGGAACAAATAGGATGGATGACAAGTAATCCACCTATCCCAGTAGGAGAAATCTATAAAAGATGGATAATCCTGGGATTAAATAAAATAGTAAGGATGTATAGCCCTACCAGCATTTTGGACATAAGACAAGGACCAAAGGAACCCTTTAGAGACTATGTAGACCGGTTCTATAAAACTCTAAGAGCCGAGCAAGCTTCACAGGAGGTAAAAAATTGGATGACAGAAACCTTGTTGGTCCAAAATGCGAACCCAGATTGTAAGACTATTTTAAAAGCATTGGGACCAGCAGCTACACTAGAAGAAATGATGACAGCATGTCAGGGAGTGGGAGGACCCAGCCATAAAGCAAGAATTTTGGCAGAAGCAATGAGCCAAGTAACAAATTCAGCTACCATAATGATGCAGAAAGGCAATTTTAGGAACCAAAGAAAGATTGTTAAATGTTTCAATTGTGGCAAAGAAGGGCACATAGCCAGAAACTGCAGGGCCCCTAGGAAAAGGGGCTGTTGGAAATGTGGAAAGGAAGGACACCAAATGAAAGACTGTACTGAGAGACAGGCTAATTTTTTAGGGAAAATCTGGCCTTCCCACAAAGGAAGGCCAGGGAATTTCCTTCAGAGCAGACCAGAGCCAACAGCCCCACCAGCCCCACCAGAAGAGAGCTTCAGGTTTGGGGAAGAGACAACAACTCCCTCTCAGAAGCAGGAGCTGATAGACAAGGAACTGTATCCTTTAGCTTCCCTCAGATCAC TCTTTGGCAACGACCCCTCGTCACAATAA70 HIV Pol; Bal ATGAATTTGCCAGGAAGATGGAAACCAAAAATGATAGGGGGAATTGGAGGTTTTATCAAAGTAAGACAGTATGATCAGATACTCATAGAAATCTGTGGACATAAAGCTATAGGTACAGTATTAATAGGACCTACACCTGTCAACATAATTGGAAGAAATCTGTTGACTCAGATTGGTTGCACTTTAAATTTTCCCATTAGTCCTATTGAAACTGTACCAGTAAAATTAAAACCAGGAATGGATGGCCCAAAAGTTAAACAATGGCCACTGACAGAAGAAAAAATAAAAGCATTAATGGAAATCTGTACAGAAATGGAAAAGGAAGGGAAAATTTCAAAAATTGGGCCTGAAAATCCATACAATACTCCAGTATTTGCCATAAAGAAAAAAGACAGTACTAAATGGAGAAAATTAGTAGATTTCAGAGAACTTAATAAGAAAACTCAAGACTTCTGGGAAGTACAATTAGGAATACACATCCCGCAGGGGTTAAAAAAGAAAAAATCAGTAACAGTACTGGATGTGGGTGATGCATATTTTTCAGTTCCCTTAGATAAAGAATTCAGGAAGTATACTGCATTTACCATACCTAGTATAAACAATGAAACACCAGGGATCAGATATCAGTACAATGTACTTCCACAGGGATGGAAAGGATCACCAGCAATATTTCAAAGTAGCATGACAAGAATCTTAGAGCCTTTTAGAAAACAAAATCCAGAAATAGTGATCTATCAATACATGGATGATTTGTATGTAGGATCTGACTTAGAAATAGGGCAGCATAGAACAAAAATAGAGGAACTGAGACAACATCTGTTGAGGTGGGGATTTACCACACCAGACAAAAAACATCAGAAAGAACCTCCATTCCTTTGGATGGGTTATGAACTCCATCCTGATAAATGGACAGTACAGCCTATAGTGCTGCCAGAAAAAGACAGCTGGACTGTCAATGACATACAGAAGTTAGTGGGAAAATTGAATTGGGCAAGTCAGATTTACCCAGGAATTAAAGTAAAGCAATTATGTAGGCTCCTTAGGGGAACCAAGGCATTAACAGAAGTAATACCACTAACAAAAGAAACAGAGCTAGAACTGGCAGAGAACAGGGAAATTCTAAAAGAACCAGTACATGGGGTGTATTATGACCCATCAAAAGACTTAATAGCAGAAATACAGAAGCAGGGGCAAGGCCAATGGACATATCAAATTTATCAAGAGCCATTTAAAAATCTGAAAACAGGAAAATATGCAAGAATGAGGGGTGCCCACACTAATGATGTAAAACAATTAACAGAGGCAGTGCAAAAAATAACCACAGAAAGCATAGTAATATGGGGAAAGACTCCTAAATTTAAACTACCCATACAAAAAGAAACATGGGAAACATGGTGGACAGAGTATTGGCAAGCCACCTGGATTCCTGAGTGGGAGTTTGTCAATACCCCTCCCTTAGTGAAATTATGGTACCAGTTAGAGAAAGAACCCATAATAGGAGCAGAAACATTCTATGTAGATGGAGCAGCTAACCGGGAGACTAAATTAGGAAAAGCAGGATATGTTACTAACAGAGGAAGACAAAAAGTTGTCTCCCTAACTGACACAACAAATCAGAAGACTGAGTTACAAGCAATTCATCTAGCTTTACAAGATTCAGGATTAGAAGTAAACATAGTAACAGACTCACAATATGCATTAGGAATCATTCAAGCACAACCAGATAAAAGTGAATCAGAGTTAGTCAGTCAAATAATAGAACAGTTAATAAAAAAGGAAAAGGTCTACCTGGCATGGGTACCAGCGCACAAAGGAATTGGAGGAAATGAACAAGTAGATAAATTAGT CAGTACTGGAATCAGGAAAGTACTA 71HIV TTTTTAGATGGAATAGATATAGCCCAAGAAGAACATGAGA Integrase; BalAATATCACAGTAATTGGAGAGCAATGGCTAGTGATTTTAACCTGCCACCTGTGGTAGCAAAAGAAATAGTAGCCAGCTGTGATAAATGTCAGCTAAAAGGAGAAGCCATGCATGGACAAGTAGACTGTAGTCCAGGAATATGGCAACTAGATTGTACACATTTAGAAGGAAAAATTATCCTGGTAGCAGTTCATGTAGCCAGTGGATATATAGAAGCAGAAGTTATTCCAGCAGAGACAGGGCAGGAAACAGCATACTTTCTCTTAAAATTAGCAGGAAGATGGCCAGTAAAAACAATACATACAGACAATGGCAGCAATTTCACTAGTACTACAGTCAAGGCCGCCTGTTGGTGGGCGGGGATCAAGCAGGAATTTGGCATTCCCTACAATCCCCAAAGTCAGGGAGTAGTAGAATCTATAAATAAAGAATTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTTAAAACAGCAGTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTATAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCACTTTGGAAAGGACCAGCAAAGCTTCTCTGGAAAGGTGAAGGGGCAGTAGTAATACAAGATAATAGTGACATAAAAGTAGTACCAAGAAGAAAAGCAAAGATCATTAGGGATTATGGAAAACAGATGGCAGGTGATGATTGTGTG GCAAGTAGACAGGATGAGGATTAG 72Envelope; ATGAAACTCCCAACAGGAATGGTCATTTTATGTAGCCTAAT RD114AATAGTTCGGGCAGGGTTTGACGACCCCCGCAAGGCTATCGCATTAGTACAAAAACAACATGGTAAACCATGCGAATGCAGCGGAGGGCAGGTATCCGAGGCCCCACCGAACTCCATCCAACAGGTAACTTGCCCAGGCAAGACGGCCTACTTAATGACCAACCAAAAATGGAAATGCAGAGTCACTCCAAAAAATCTCACCCCTAGCGGGGGAGAACTCCAGAACTGCCCCTGTAACACTTTCCAGGACTCGATGCACAGTTCTTGTTATACTGAATACCGGCAATGCAGGGCGAATAATAAGACATACTACACGGCCACCTTGCTTAAAATACGGTCTGGGAGCCTCAACGAGGTACAGATATTACAAAACCCCAATCAGCTCCTACAGTCCCCTTGTAGGGGCTCTATAAATCAGCCCGTTTGCTGGAGTGCCACAGCCCCCATCCATATCTCCGATGGTGGAGGACCCCTCGATACTAAGAGAGTGTGGACAGTCCAAAAAAGGCTAGAACAAATTCATAAGGCTATGCATCCTGAACTTCAATACCACCCCTTAGCCCTGCCCAAAGTCAGAGATGACCTTAGCCTTGATGCACGGACTTTTGATATCCTGAATACCACTTTTAGGTTACTCCAGATGTCCAATTTTAGCCTTGCCCAAGATTGTTGGCTCTGTTTAAAACTAGGTACCCCTACCCCTCTTGCGATACCCACTCCCTCTTTAACCTACTCCCTAGCAGACTCCCTAGCGAATGCCTCCTGTCAGATTATACCTCCCCTCTTGGTTCAACCGATGCAGTTCTCCAACTCGTCCTGTTTATCTTCCCCTTTCATTAACGATACGGAACAAATAGACTTAGGTGCAGTCACCTTTACTAACTGCACCTCTGTAGCCAATGTCAGTAGTCCTTTATGTGCCCTAAACGGGTCAGTCTTCCTCTGTGGAAATAACATGGCATACACCTATTTACCCCAAAACTGGACAGGACTTTGCGTCCAAGCCTCCCTCCTCCCCGACATTGACATCATCCCGGGGGATGAGCCAGTCCCCATTCCTGCCATTGATCATTATATACATAGACCTAAACGAGCTGTACAGTTCATCCCTTTACTAGCTGGACTGGGAATCACCGCAGCATTCACCACCGGAGCTACAGGCCTAGGTGTCTCCGTCACCCAGTATACAAAATTATCCCATCAGTTAATATCTGATGTCCAAGTCTTATCCGGTACCATACAAGATTTACAAGACCAGGTAGACTCGTTAGCTGAAGTAGTTCTCCAAAATAGGAGGGGACTGGACCTACTAACGGCAGAACAAGGAGGAATTTGTTTAGCCTTACAAGAAAAATGCTGTTTTTATGCTAACAAGTCAGGAATTGTGAGAAACAAAATAAGAACCCTACAAGAAGAATTACAAAAACGCAGGGAAAGCCTGGCATCCAACCCTCTCTGGACCGGGCTGCAGGGCTTTCTTCCGTACCTCCTACCTCTCCTGGGACCCCTACTCACCCTCCTACTCATACTAACCATTGGGCCATGCGTTTTCAATCGATTGGTCCAATTTGTTAAAGACAGGATCTCAGTGGTCCAGGCTCTGGTTTTGACTCAGCAATATCACCAGCTA AAACCCATAGAGTACGAGCCATGA 73Envelope; ATGCTTCTCACCTCAAGCCCGCACCACCTTCGGCACCAGAT GALVGAGTCCTGGGAGCTGGAAAAGACTGATCATCCTCTTAAGCTGCGTATTCGGAGACGGCAAAACGAGTCTGCAGAATAAGAACCCCCACCAGCCTGTGACCCTCACCTGGCAGGTACTGTCCCAAACTGGGGACGTTGTCTGGGACAAAAAGGCAGTCCAGCCCCTTTGGACTTGGTGGCCCTCTCTTACACCTGATGTATGTGCCCTGGCGGCCGGTCTTGAGTCCTGGGATATCCCGGGATCCGATGTATCGTCCTCTAAAAGAGTTAGACCTCCTGATTCAGACTATACTGCCGCTTATAAGCAAATCACCTGGGGAGCCATAGGGTGCAGCTACCCTCGGGCTAGGACCAGGATGGCAAATTCCCCCTTCTACGTGTGTCCCCGAGCTGGCCGAACCCATTCAGAAGCTAGGAGGTGTGGGGGGCTAGAATCCCTATACTGTAAAGAATGGAGTTGTGAGACCACGGGTACCGTTTATTGGCAACCCAAGTCCTCATGGGACCTCATAACTGTAAAATGGGACCAAAATGTGAAATGGGAGCAAAAATTTCAAAAGTGTGAACAAACCGGCTGGTGTAACCCCCTCAAGATAGACTTCACAGAAAAAGGAAAACTCTCCAGAGATTGGATAACGGAAAAAACCTGGGAATTAAGGTTCTATGTATATGGACACCCAGGCATACAGTTGACTATCCGCTTAGAGGTCACTAACATGCCGGTTGTGGCAGTGGGCCCAGACCCTGTCCTTGCGGAACAGGGACCTCCTAGCAAGCCCCTCACTCTCCCTCTCTCCCCACGGAAAGCGCCGCCCACCCCTCTACCCCCGGCGGCTAGTGAGCAAACCCCTGCGGTGCATGGAGAAACTGTTACCCTAAACTCTCCGCCTCCCACCAGTGGCGACCGACTCTTTGGCCTTGTGCAGGGGGCCTTCCTAACCTTGAATGCTACCAACCCAGGGGCCACTAAGTCTTGCTGGCTCTGTTTGGGCATGAGCCCCCCTTATTATGAAGGGATAGCCTCTTCAGGAGAGGTCGCTTATACCTCCAACCATACCCGATGCCACTGGGGGGCCCAAGGAAAGCTTACCCTCACTGAGGTCTCCGGACTCGGGTCATGCATAGGGAAGGTGCCTCTTACCCATCAACATCTTTGCAACCAGACCTTACCCATCAATTCCTCTAAAAACCATCAGTATCTGCTCCCCTCAAACCATAGCTGGTGGGCCTGCAGCACTGGCCTCACCCCCTGCCTCTCCACCTCAGTTTTTAATCAGTCTAAAGACTTCTGTGTCCAGGTCCAGCTGATCCCCCGCATCTATTACCATTCTGAAGAAACCTTGTTACAAGCCTATGACAAATCACCCCCCAGGTTTAAAAGAGAGCCTGCCTCACTTACCCTAGCTGTCTTCCTGGGGTTAGGGATTGCGGCAGGTATAGGTACTGGCTCAACCGCCCTAATTAAAGGGCCCATAGACCTCCAGCAAGGCCTAACCAGCCTCCAAATCGCCATTGACGCTGACCTCCGGGCCCTTCAGGACTCAATCAGCAAGCTAGAGGACTCACTGACTTCCCTATCTGAGGTAGTACTCCAAAATAGGAGAGGCCTTGACTTACTATTCCTTAAAGAAGGAGGCCTCTGCGCGGCCCTAAAAGAAGAGTGCTGTTTTTATGTAGACCACTCAGGTGCAGTACGAGACTCCATGAAAAAACTTAAAGAAAGACTAGATAAAAGACAGTTAGAGCGCCAGAAAAACCAAAACTGGTATGAAGGGTGGTTCAATAACTCCCCTTGGTTTACTACCCTACTATCAACCATCGCTGGGCCCCTATTGCTCCTCCTTTTGTTACTCACTCTTGGGCCCTGCATCATCAATAAATTAATCCAATTCATCAATGATAGGATAAGTGCAGTCAAAATTTTAGTCCTTAGACAGAAATATCAGACCCTAG ATAACGAGGAAAACCTTTAA 74Envelope; ATGGTTCCGCAGGTTCTTTTGTTTGTACTCCTTCTGGGTTTT FUGTCGTTGTGTTTCGGGAAGTTCCCCATTTACACGATACCAGACGAACTTGGTCCCTGGAGCCCTATTGACATACACCATCTCAGCTGTCCAAATAACCTGGTTGTGGAGGATGAAGGATGTACCAACCTGTCCGAGTTCTCCTACATGGAACTCAAAGTGGGATACATCTCAGCCATCAAAGTGAACGGGTTCACTTGCACAGGTGTTGTGACAGAGGCAGAGACCTACACCAACTTTGTTGGTTATGTCACAACCACATTCAAGAGAAAGCATTTCCGCCCCACCCCAGACGCATGTAGAGCCGCGTATAACTGGAAGATGGCCGGTGACCCCAGATATGAAGAGTCCCTACACAATCCATACCCCGACTACCACTGGCTTCGAACTGTAAGAACCACCAAAGAGTCCCTCATTATCATATCCCCAAGTGTGACAGATTTGGACCCATATGACAAATCCCTTCACTCAAGGGTCTTCCCTGGCGGAAAGTGCTCAGGAATAACGGTGTCCTCTACCTACTGCTCAACTAACCATGATTACACCATTTGGATGCCCGAGAATCCGAGACCAAGGACACCTTGTGACATTTTTACCAATAGCAGAGGGAAGAGAGCATCCAACGGGAACAAGACTTGCGGCTTTGTGGATGAAAGAGGCCTGTATAAGTCTCTAAAAGGAGCATGCAGGCTCAAGTTATGTGGAGTTCTTGGACTTAGACTTATGGATGGAACATGGGTCGCGATGCAAACATCAGATGAGACCAAATGGTGCCCTCCAGATCAGTTGGTGAATTTGCACGACTTTCGCTCAGACGAGATCGAGCATCTCGTTGTGGAGGAGTTAGTTAAGAAAAGAGAGGAATGTCTGGATGCATTAGAGTCCATCATGACCACCAAGTCAGTAAGTTTCAGACGTCTCAGTCACCTGAGAAAACTTGTCCCAGGGTTTGGAAAAGCATATACCATATTCAACAAAACCTTGATGGAGGCTGATGCTCACTACAAGTCAGTCCGGACCTGGAATGAGATCATCCCCTCAAAAGGGTGTTTGAAAGTTGGAGGAAGGTGCCATCCTCATGTGAACGGGGTGTTTTTCAATGGTATAATATTAGGGCCTGACGACCATGTCCTAATCCCAGAGATGCAATCATCCCTCCTCCAGCAACATATGGAGTTGTTGGAATCTTCAGTTATCCCCCTGATGCACCCCCTGGCAGACCCTTCTACAGTTTTCAAAGAAGGTGATGAGGCTGAGGATTTTGTTGAAGTTCACCTCCCCGATGTGTACAAACAGATCTCAGGGGTTGACCTGGGTCTCCCGAACTGGGGAAAGTATGTATTGATGACTGCAGGGGCCATGATTGGCCTGGTGTTGATATTTTCCCTAATGACATGGTGCAGAGTTGGTATCCATCTTTGCATTAAATTAAAGCACACCAAGAAAAGACAGATTTATACAGACA TAGAGATGAACCGACTTGGAAAGTAA 75Envelope; ATGGGTCAGATTGTGACAATGTTTGAGGCTCTGCCTCACAT LCMVCATCGATGAGGTGATCAACATTGTCATTATTGTGCTTATCGTGATCACGGGTATCAAGGCTGTCTACAATTTTGCCACCTGTGGGATATTCGCATTGATCAGTTTCCTACTTCTGGCTGGCAGGTCCTGTGGCATGTACGGTCTTAAGGGACCCGACATTTACAAAGGAGTTTACCAATTTAAGTCAGTGGAGTTTGATATGTCACATCTGAACCTGACCATGCCCAACGCATGTTCAGCCAACAACTCCCACCATTACATCAGTATGGGGACTTCTGGACTAGAATTGACCTTCACCAATGATTCCATCATCAGTCACAACTTTTGCAATCTGACCTCTGCCTTCAACAAAAAGACCTTTGACCACACACTCATGAGTATAGTTTCGAGCCTACACCTCAGTATCAGAGGGAACTCCAACTATAAGGCAGTATCCTGCGACTTCAACAATGGCATAACCATCCAATACAACTTGACATTCTCAGATCGACAAAGTGCTCAGAGCCAGTGTAGAACCTTCAGAGGTAGAGTCCTAGATATGTTTAGAACTGCCTTCGGGGGGAAATACATGAGGAGTGGCTGGGGCTGGACAGGCTCAGATGGCAAGACCACCTGGTGTAGCCAGACGAGTTACCAATACCTGATTATACAAAATAGAACCTGGGAAAACCACTGCACATATGCAGGTCCTTTTGGGATGTCCAGGATTCTCCTTTCCCAAGAGAAGACTAAGTTCTTCACTAGGAGACTAGCGGGCACATTCACCTGGACTTTGTCAGACTCTTCAGGGGTGGAGAATCCAGGTGGTTATTGCCTGACCAAATGGATGATTCTTGCTGCAGAGCTTAAGTGTTTCGGGAACACAGCAGTTGCGAAATGCAATGTAAATCATGATGCCGAATTCTGTGACATGCTGCGACTAATTGACTACAACAAGGCTGCTTTGAGTAAGTTCAAAGAGGACGTAGAATCTGCCTTGCACTTATTCAAAACAACAGTGAATTCTTTGATTTCAGATCAACTACTGATGAGGAACCACTTGAGAGATCTGATGGGGGTGCCATATTGCAATTACTCAAAGTTTTGGTACCTAGAACATGCAAAGACCGGCGAAACTAGTGTCCCCAAGTGCTGGCTTGTCACCAATGGTTCTTACTTAAATGAGACCCACTTCAGTGATCAAATCGAACAGGAAGCCGATAACATGATTACAGAGATGTTGAGGAAGGATTACATAAAGAGGCAGGGGAGTACCCCCCTAGCATTGATGGACCTTCTGATGTTTTCCACATCTGCATATCTAGTCAGCATCTTCCTGCACCTTGTCAAAATACCAACACACAGGCACATAAAAGGTGGCTCATGTCCAAAGCCACACCGATTAACCAACAAAGGAATTTGTAGTTGTGGTGCATTTAAGGTGCCTG GTGTAAAAACCGTCTGGAAAAGACGCTGA76 Envelope; ATGAACACTCAAATCCTGGTTTTCGCCCTTGTGGCAGTCAT FPVCCCCACAAATGCAGACAAAATTTGTCTTGGACATCATGCTGTATCAAATGGCACCAAAGTAAACACACTCACTGAGAGAGGAGTAGAAGTTGTCAATGCAACGGAAACAGTGGAGCGGACAAACATCCCCAAAATTTGCTCAAAAGGGAAAAGAACCACTGATCTTGGCCAATGCGGACTGTTAGGGACCATTACCGGACCACCTCAATGCGACCAATTTCTAGAATTTTCAGCTGATCTAATAATCGAGAGACGAGAAGGAAATGATGTTTGTTACCCGGGGAAGTTTGTTAATGAAGAGGCATTGCGACAAATCCTCAGAGGATCAGGTGGGATTGACAAAGAAACAATGGGATTCACATATAGTGGAATAAGGACCAACGGAACAACTAGTGCATGTAGAAGATCAGGGTCTTCATTCTATGCAGAAATGGAGTGGCTCCTGTCAAATACAGACAATGCTGCTTTCCCACAAATGACAAAATCATACAAAAACACAAGGAGAGAATCAGCTCTGATAGTCTGGGGAATCCACCATTCAGGATCAACCACCGAACAGACCAAACTATATGGGAGTGGAAATAAACTGATAACAGTCGGGAGTTCCAAATATCATCAATCTTTTGTGCCGAGTCCAGGAACACGACCGCAGATAAATGGCCAGTCCGGACGGATTGATTTTCATTGGTTGATCTTGGATCCCAATGATACAGTTACTTTTAGTTTCAATGGGGCTTTCATAGCTCCAAATCGTGCCAGCTTCTTGAGGGGAAAGTCCATGGGGATCCAGAGCGATGTGCAGGTTGATGCCAATTGCGAAGGGGAATGCTACCACAGTGGAGGGACTATAACAAGCAGATTGCCTTTTCAAAACATCAATAGCAGAGCAGTTGGCAAATGCCCAAGATATGTAAAACAGGAAAGTTTATTATTGGCAACTGGGATGAAGAACGTTCCCGAACCTTCCAAAAAAAGGAAAAAAAGAGGCCTGTTTGGCGCTATAGCAGGGTTTATTGAAAATGGTTGGGAAGGTCTGGTCGACGGGTGGTACGGTTTCAGGCATCAGAATGCACAAGGAGAAGGAACTGCAGCAGACTACAAAAGCACCCAATCGGCAATTGATCAGATAACCGGAAAGTTAAATAGACTCATTGAGAAAACCAACCAGCAATTTGAGCTAATAGATAATGAATTCACTGAGGTGGAAAAGCAGATTGGCAATTTAATTAACTGGACCAAAGACTCCATCACAGAAGTATGGTCTTACAATGCTGAACTTCTTGTGGCAATGGAAAACCAGCACACTATTGATTTGGCTGATTCAGAGATGAACAAGCTGTATGAGCGAGTGAGGAAACAATTAAGGGAAAATGCTGAAGAGGATGGCACTGGTTGCTTTGAAATTTTTCATAAATGTGACGATGATTGTATGGCTAGTATAAGGAACAATACTTATGATCACAGCAAATACAGAGAAGAAGCGATGCAAAATAGAATACAAATTGACCCAGTCAAATTGAGTAGTGGCTACAAAGATGTGATACTTTGGTTTAGCTTCGGGGCATCATGCTTTTTGCTTCTTGCCATTGCAATGGGCCTTGTTTTCATATGTGTGAAGAACGGAAACATGCGGTGCACTATTTGTATATAA 77 Envelope;AGTGTAACAGAGCACTTTAATGTGTATAAGGCTACTAGACC RRVATACCTAGCACATTGCGCCGATTGCGGGGACGGGTACTTCTGCTATAGCCCAGTTGCTATCGAGGAGATCCGAGATGAGGCGTCTGATGGCATGCTTAAGATCCAAGTCTCCGCCCAAATAGGTCTGGACAAGGCAGGCACCCACGCCCACACGAAGCTCCGATATATGGCTGGTCATGATGTTCAGGAATCTAAGAGAGATTCCTTGAGGGTGTACACGTCCGCAGCGTGCTCCATACATGGGACGATGGGACACTTCATCGTCGCACACTGTCCACCAGGCGACTACCTCAAGGTTTCGTTCGAGGACGCAGATTCGCACGTGAAGGCATGTAAGGTCCAATACAAGCACAATCCATTGCCGGTGGGTAGAGAGAAGTTCGTGGTTAGACCACACTTTGGCGTAGAGCTGCCATGCACCTCATACCAGCTGACAACGGCTCCCACCGACGAGGAGATTGACATGCATACACCGCCAGATATACCGGATCGCACCCTGCTATCACAGACGGCGGGCAACGTCAAAATAACAGCAGGCGGCAGGACTATCAGGTACAACTGTACCTGCGGCCGTGACAACGTAGGCACTACCAGTACTGACAAGACCATCAACACATGCAAGATTGACCAATGCCATGCTGCCGTCACCAGCCATGACAAATGGCAATTTACCTCTCCATTTGTTCCCAGGGCTGATCAGACAGCTAGGAAAGGCAAGGTACACGTTCCGTTCCCTCTGACTAACGTCACCTGCCGAGTGCCGTTGGCTCGAGCGCCGGATGCCACCTATGGTAAGAAGGAGGTGACCCTGAGATTACACCCAGATCATCCGACGCTCTTCTCCTATAGGAGTTTAGGAGCCGAACCGCACCCGTACGAGGAATGGGTTGACAAGTTCTCTGAGCGCATCATCCCAGTGACGGAAGAAGGGATTGAGTACCAGTGGGGCAACAACCCGCCGGTCTGCCTGTGGGCGCAACTGACGACCGAGGGCAAACCCCATGGCTGGCCACATGAAATCATTCAGTACTATTATGGACTATACCCCGCCGCCACTATTGCCGCAGTATCCGGGGCGAGTCTGATGGCCCTCCTAACTCTGGCGGCCACATGCTGCATGCTGGCCACCGCGAGGAGAAAGTGCCTAACACCGTACGCCCTGACGCCAGGAGCGGTGGTACCGTTGACACTGGGGCTGCTTTGCTGCGCACCG AGGGCGAATGCA 78 Envelope;AGTGTAACAGAGCACTTTAATGTGTATAAGGCTACTAGACC MLV 10A1ATACCTAGCACATTGCGCCGATTGCGGGGACGGGTACTTCTGCTATAGCCCAGTTGCTATCGAGGAGATCCGAGATGAGGCGTCTGATGGCATGCTTAAGATCCAAGTCTCCGCCCAAATAGGTCTGGACAAGGCAGGCACCCACGCCCACACGAAGCTCCGATATATGGCTGGTCATGATGTTCAGGAATCTAAGAGAGATTCCTTGAGGGTGTACACGTCCGCAGCGTGCTCCATACATGGGACGATGGGACACTTCATCGTCGCACACTGTCCACCAGGCGACTACCTCAAGGTTTCGTTCGAGGACGCAGATTCGCACGTGAAGGCATGTAAGGTCCAATACAAGCACAATCCATTGCCGGTGGGTAGAGAGAAGTTCGTGGTTAGACCACACTTTGGCGTAGAGCTGCCATGCACCTCATACCAGCTGACAACGGCTCCCACCGACGAGGAGATTGACATGCATACACCGCCAGATATACCGGATCGCACCCTGCTATCACAGACGGCGGGCAACGTCAAAATAACAGCAGGCGGCAGGACTATCAGGTACAACTGTACCTGCGGCCGTGACAACGTAGGCACTACCAGTACTGACAAGACCATCAACACATGCAAGATTGACCAATGCCATGCTGCCGTCACCAGCCATGACAAATGGCAATTTACCTCTCCATTTGTTCCCAGGGCTGATCAGACAGCTAGGAAAGGCAAGGTACACGTTCCGTTCCCTCTGACTAACGTCACCTGCCGAGTGCCGTTGGCTCGAGCGCCGGATGCCACCTATGGTAAGAAGGAGGTGACCCTGAGATTACACCCAGATCATCCGACGCTCTTCTCCTATAGGAGTTTAGGAGCCGAACCGCACCCGTACGAGGAATGGGTTGACAAGTTCTCTGAGCGCATCATCCCAGTGACGGAAGAAGGGATTGAGTACCAGTGGGGCAACAACCCGCCGGTCTGCCTGTGGGCGCAACTGACGACCGAGGGCAAACCCCATGGCTGGCCACATGAAATCATTCAGTACTATTATGGACTATACCCCGCCGCCACTATTGCCGCAGTATCCGGGGCGAGTCTGATGGCCCTCCTAACTCTGGCGGCCACATGCTGCATGCTGGCCACCGCGAGGAGAAAGTGCCTAACACCGTACGCCCTGACGCCAGGAGCGGTGGTACCGTTGACACTGGGGCTGCTTTGCTGCGCACCG AGGGCGAATGCA 79 Envelope;ATGGGTGTTACAGGAATATTGCAGTTACCTCGTGATCGATT EbolaCAAGAGGACATCATTCTTTCTTTGGGTAATTATCCTTTTCCAAAGAACATTTTCCATCCCACTTGGAGTCATCCACAATAGCACATTACAGGTTAGTGATGTCGACAAACTGGTTTGCCGTGACAAACTGTCATCCACAAATCAATTGAGATCAGTTGGACTGAATCTCGAAGGGAATGGAGTGGCAACTGACGTGCCATCTGCAACTAAAAGATGGGGCTTCAGGTCCGGTGTCCCACCAAAGGTGGTCAATTATGAAGCTGGTGAATGGGCTGAAAACTGCTACAATCTTGAAATCAAAAAACCTGACGGGAGTGAGTGTCTACCAGCAGCGCCAGACGGGATTCGGGGCTTCCCCCGGTGCCGGTATGTGCACAAAGTATCAGGAACGGGACCGTGTGCCGGAGACTTTGCCTTCCACAAAGAGGGTGCTTTCTTCCTGTATGACCGACTTGCTTCCACAGTTATCTACCGAGGAACGACTTTCGCTGAAGGTGTCGTTGCATTTCTGATACTGCCCCAAGCTAAGAAGGACTTCTTCAGCTCACACCCCTTGAGAGAGCCGGTCAATGCAACGGAGGACCCGTCTAGTGGCTACTATTCTACCACAATTAGATATCAAGCTACCGGTTTTGGAACCAATGAGACAGAGTATTTGTTCGAGGTTGACAATTTGACCTACGTCCAACTTGAATCAAGATTCACACCACAGTTTCTGCTCCAGCTGAATGAGACAATATATACAAGTGGGAAAAGGAGCAATACCACGGGAAAACTAATTTGGAAGGTCAACCCCGAAATTGATACAACAATCGGGGAGTGGGCCTTCTGGGAAACTAAAAAAACCTCACTAGAAAAATTCGCAGTGAAGAGTTGTCTTTCACAGCTGTATCAAACAGAGCCAAAAACATCAGTGGTCAGAGTCCGGCGCGAACTTCTTCCGACCCAGGGACCAACACAACAACTGAAGACCACAAAATCATGGCTTCAGAAAATTCCTCTGCAATGGTTCAAGTGCACAGTCAAGGAAGGGAAGCTGCAGTGTCGCATCTGACAACCCTTGCCACAATCTCCACGAGTCCTCAACCCCCCACAACCAAACCAGGTCCGGACAACAGCACCCACAATACACCCGTGTATAAACTTGACATCTCTGAGGCAACTCAAGTTGAACAACATCACCGCAGAACAGACAACGACAGCACAGCCTCCGACACTCCCCCCGCCACGACCGCAGCCGGACCCCTAAAAGCAGAGAACACCAACACGAGCAAGGGTACCGACCTCCTGGACCCCGCCACCACAACAAGTCCCCAAAACCACAGCGAGACCGCTGGCAACAACAACACTCATCACCAAGATACCGGAGAAGAGAGTGCCAGCAGCGGGAAGCTAGGCTTAATTACCAATACTATTGCTGGAGTCGCAGGACTGATCACAGGCGGGAGGAGAGCTCGAAGAGAAGCAATTGTCAATGCTCAACCCAAATGCAACCCTAATTTACATTACTGGACTACTCAGGATGAAGGTGCTGCAATCGGACTGGCCTGGATACCATATTTCGGGCCAGCAGCCGAGGGAATTTACATAGAGGGGCTGATGCACAATCAAGATGGTTTAATCTGTGGGTTGAGACAGCTGGCCAACGAGACGACTCAAGCTCTTCAACTGTTCCTGAGAGCCACAACCGAGCTACGCACCTTTTCAATCCTCAACCGTAAGGCAATTGATTTCTTGCTGCAGCGATGGGGCGGCACATGCCACATTTTGGGACCGGACTGCTGTATCGAACCACATGATTGGACCAAGAACATAACAGACAAAATTGATCAGATTATTCATGATTTTGTTGATAAAACCCTTCCGGACCAGGGGGACAATGACAATTGGTGGACAGGATGGAGACAATGGATACCGGCAGGTATTGGAGTTACAGGCGTTATAATTGCAGTTATCGCTTTATTCTGTATATGCAAATTTGT CTTTTAG 80 Short WPREAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGA sequenceTATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTAATGCCTCTGTATCATGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATA ATTCCGTGGTGTTGTC 81 PrimerTAAGCAGAATTCATGAATTTGCCAGGAAGAT 82 PrimerCCATACAATGAATGGACACTAGGCGGCCGCACGAAT 83 Gag, Pol,GAATTCATGAATTTGCCAGGAAGATGGAAACCAAAAATGA IntegraseTAGGGGGAATTGGAGGTTTTATCAAAGTAAGACAGTATGA fragmentTCAGATACTCATAGAAATCTGCGGACATAAAGCTATAGGTACAGTATTAGTAGGACCTACACCTGTCAACATAATTGGAAGAAATCTGTTGACTCAGATTGGCTGCACTTTAAATTTTCCCATTAGTCCTATTGAGACTGTACCAGTAAAATTAAAGCCAGGAATGGATGGCCCAAAAGTTAAACAATGGCCATTGACAGAAGAAAAAATAAAAGCATTAGTAGAAATTTGTACAGAAATGGAAAAGGAAGGAAAAATTTCAAAAATTGGGCCTGAAAATCCATACAATACTCCAGTATTTGCCATAAAGAAAAAAGACAGTACTAAATGGAGAAAATTAGTAGATTTCAGAGAACTTAATAAGAGAACTCAAGATTTCTGGGAAGTTCAATTAGGAATACCACATCCTGCAGGGTTAAAACAGAAAAAATCAGTAACAGTACTGGATGTGGGCGATGCATATTTTTCAGTTCCCTTAGATAAAGACTTCAGGAAGTATACTGCATTTACCATACCTAGTATAAACAATGAGACACCAGGGATTAGATATCAGTACAATGTGCTTCCACAGGGATGGAAAGGATCACCAGCAATATTCCAGTGTAGCATGACAAAAATCTTAGAGCCTTTTAGAAAACAAAATCCAGACATAGTCATCTATCAATACATGGATGATTTGTATGTAGGATCTGACTTAGAAATAGGGCAGCATAGAACAAAAATAGAGGAACTGAGACAACATCTGTTGAGGTGGGGATTTACCACACCAGACAAAAAACATCAGAAAGAACCTCCATTCCTTTGGATGGGTTATGAACTCCATCCTGATAAATGGACAGTACAGCCTATAGTGCTGCCAGAAAAGGACAGCTGGACTGTCAATGACATACAGAAATTAGTGGGAAAATTGAATTGGGCAAGTCAGATTTATGCAGGGATTAAAGTAAGGCAATTATGTAAACTTCTTAGGGGAACCAAAGCACTAACAGAAGTAGTACCACTAACAGAAGAAGCAGAGCTAGAACTGGCAGAAAACAGGGAGATTCTAAAAGAACCGGTACATGGAGTGTATTATGACCCATCAAAAGACTTAATAGCAGAAATACAGAAGCAGGGGCAAGGCCAATGGACATATCAAATTTATCAAGAGCCATTTAAAAATCTGAAAACAGGAAAGTATGCAAGAATGAAGGGTGCCCACACTAATGATGTGAAACAATTAACAGAGGCAGTACAAAAAATAGCCACAGAAAGCATAGTAATATGGGGAAAGACTCCTAAATTTAAATTACCCATACAAAAGGAAACATGGGAAGCATGGTGGACAGAGTATTGGCAAGCCACCTGGATTCCTGAGTGGGAGTTTGTCAATACCCCTCCCTTAGTGAAGTTATGGTACCAGTTAGAGAAAGAACCCATAATAGGAGCAGAAACTTTCTATGTAGATGGGGCAGCCAATAGGGAAACTAAATTAGGAAAAGCAGGATATGTAACTGACAGAGGAAGACAAAAAGTTGTCCCCCTAACGGACACAACAAATCAGAAGACTGAGTTACAAGCAATTCATCTAGCTTTGCAGGATTCGGGATTAGAAGTAAACATAGTGACAGACTCACAATATGCATTGGGAATCATTCAAGCACAACCAGATAAGAGTGAATCAGAGTTAGTCAGTCAAATAATAGAGCAGTTAATAAAAAAGGAAAAAGTCTACCTGGCATGGGTACCAGCACACAAAGGAATTGGAGGAAATGAACAAGTAGATAAATTGGTCAGTGCTGGAATCAGGAAAGTACTATTTTTAGATGGAATAGATAAGGCCCAAGAAGAACATGAGAAATATCACAGTAATTGGAGAGCAATGGCTAGTGATTTTAACCTACCACCTGTAGTAGCAAAAGAAATAGTAGCCAGCTGTGATAAATGTCAGCTAAAAGGGGAAGCCATGCATGGACAAGTAGACTGTAGCCCAGGAATATGGCAGCTAGATTGTACACATTTAGAAGGAAAAGTTATCTTGGTAGCAGTTCATGTAGCCAGTGGATATATAGAAGCAGAAGTAATTCCAGCAGAGACAGGGCAAGAAACAGCATACTTCCTCTTAAAATTAGCAGGAAGATGGCCAGTAAAAACAGTACATACAGACAATGGCAGCAATTTCACCAGTACTACAGTTAAGGCCGCCTGTTGGTGGGCGGGGATCAAGCAGGAATTTGGCATTCCCTACAATCCCCAAAGTCAAGGAGTAATAGAATCTATGAATAAAGAATTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTTAAGACAGCAGTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTTGGAAAGGACCAGCAAAGCTCCTCTGGAAAGGTGAAGGGGCAGTAGTAATACAAGATAATAGTGACATAAAAGTAGTGCCAAGAAGAAAAGCAAAGATCATCAGGGATTATGGAAAACAGATGGCAGGTGATGATTGTGTGGCAAGTAGACA GGATGAGGATTAA 84 DNATCTAGAATGGCAGGAAGAAGCGGAGACAGCGACGAAGAG FragmentCTCATCAGAACAGTCAGACTCATCAAGCTTCTCTATCAAAG containingCAACCCACCTCCCAATCCCGAGGGGACCCGACAGGCCCGA Rev, RRE andAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAG rabbit betaATCCATTCGATTAGTGAACGGATCCTTGGCACTTATCTGGG globin poly AACGATCTGCGGAGCCTGTGCCTCTTCAGCTACCACCGCTTGAGAGACTTACTCTTGATTGTAACGAGGATTGTGGAACTTCTGGGACGCAGGGGGTGGGAAGCCCTCAAATATTGGTGGAATCTCCTACAATATTGGAGTCAGGAGCTAAAGAATAGAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCATATGCTGGCTGCCATGAACAAAGGTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGAAGATCCCTCGACCTGCAGCCCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCGGATCCGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCAGCGGCCGCCCCGGG 85 DNAACGCGTTAGTTATTAATAGTAATCAATTACGGGGTCATTAG fragmentTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACG containing theGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCC CAGCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCA enhancer/ATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACG promoter/intronGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGC sequenceCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTCGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTAAAGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGGCGGTCGGGCTGTAACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTGCGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCATCTCCAGCCTCGGGGCTGCCGCAGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGGAATTC 86 DNAGAATTCATGAAGTGCCTTTTGTACTTAGCCTTTTTATTCATT fragmentGGGGTGAATTGCAAGTTCACCATAGTTTTTCCACACAACCA containingAAAAGGAAACTGGAAAAATGTTCCTTCTAATTACCATTATT VSV-GGCCCGTCAAGCTCAGATTTAAATTGGCATAATGACTTAATAGGCACAGCCTTACAAGTCAAAATGCCCAAGAGTCACAAGGCTATTCAAGCAGACGGTTGGATGTGTCATGCTTCCAAATGGGTCACTACTTGTGATTTCCGCTGGTATGGACCGAAGTATATAACACATTCCATCCGATCCTTCACTCCATCTGTAGAACAATGCAAGGAAAGCATTGAACAAACGAAACAAGGAACTTGGCTGAATCCAGGCTTCCCTCCTCAAAGTTGTGGATATGCAACTGTGACGGATGCCGAAGCAGTGATTGTCCAGGTGACTCCTCACCATGTGCTGGTTGATGAATACACAGGAGAATGGGTTGATTCACAGTTCATCAACGGAAAATGCAGCAATTACATATGCCCCACTGTCCATAACTCTACAACCTGGCATTCTGACTATAAGGTCAAAGGGCTATGTGATTCTAACCTCATTTCCATGGACATCACCTTCTTCTCAGAGGACGGAGAGCTATCATCCCTGGGAAAGGAGGGCACAGGGTTCAGAAGTAACTACTTTGCTTATGAAACTGGAGGCAAGGCCTGCAAAATGCAATACTGCAAGCATTGGGGAGTCAGACTCCCATCAGGTGTCTGGTTCGAGATGGCTGATAAGGATCTCTTTGCTGCAGCCAGATTCCCTGAATGCCCAGAAGGGTCAAGTATCTCTGCTCCATCTCAGACCTCAGTGGATGTAAGTCTAATTCAGGACGTTGAGAGGATCTTGGATTATTCCCTCTGCCAAGAAACCTGGAGCAAAATCAGAGCGGGTCTTCCAATCTCTCCAGTGGATCTCAGCTATCTTGCTCCTAAAAACCCAGGAACCGGTCCTGCTTTCACCATAATCAATGGTACCCTAAAATACTTTGAGACCAGATACATCAGAGTCGATATTGCTGCTCCAATCCTCTCAAGAATGGTCGGAATGATCAGTGGAACTACCACAGAAAGGGAACTGTGGGATGACTGGGCACCATATGAAGACGTGGAAATTGGACCCAATGGAGTTCTGAGGACCAGTTCAGGATATAAGTTTCCTTTATACATGATTGGACATGGTATGTTGGACTCCGATCTTCATCTTAGCTCAAAGGCTCAGGTGTTCGAACATCCTCACATTCAAGACGCTGCTTCGCAACTTCCTGATGATGAGAGTTTATTTTTTGGTGATACTGGGCTATCCAAAAATCCAATCGAGCTTGTAGAAGGTTGGTTCAGTAGTTGGAAAAGCTCTATTGCCTCTTTTTTCTTTATCATAGGGTTAATCATTGGACTATTCTTGGTTCTCCGAGTTGGTATCCATCTTTGCATTAAATTAAAGCACACCAAGAAAAGACAGATTTATAC AGACATAGAGATGAGAATTC 87 HelperTCTAGAAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAG plasmidGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACA containingGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAAC RRE andAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCA rabbit betaACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATC globin poly ACTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCATATGCTGGCTGCCATGAACAAAGGTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGAAGATCCCTCGACCTGCAGCCCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCGGATCCGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCACCCGG G 88 RSVCAATTGCGATGTACGGGCCAGATATACGCGTATCTGAGGG promoter andGACTAGGGTGTGTTTAGGCGAAAAGCGGGGCTTCGGTTGT HIV RevACGCGGTTAGGAGTCCCCTCAGGATATAGTAGTTTCGCTTTTGCATAGGGAGGGGGAAATGTAGTCTTATGCAATACACTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACAGGTCTGACATGGATTGGACGAACCACTGAATTCCGCATTGCAGAGATAATTGTATTTAAGTGCCTAGCTCGATACAATAAACGCCATTTGACCATTCACCACATTGGTGTGCACCTCCAAGCTCGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCCCTCGAAGCTAGCGATTAGGCATCTCCTATGGCAGGAAGAAGCGGAGACAGCGACGAAGAACTCCTCAAGGCAGTCAGACTCATCAAGTTTCTCTATCAAAGCAACCCACCTCCCAATCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCCTTAGCACTTATCTGGGACGATCTGCGGAGCCTGTGCCTCTTCAGCTACCACCGCTTGAGAGACTTACTCTTGATTGTAACGAGGATTGTGGAACTTCTGGGACGCAGGGGGTGGGAAGCCCTCAAATATTGGTGGAATCTCCTACAATATTGGAGTCAGGAGCTAAAGAATAGTCTAGA 89 Target ATGGCAGGAAGAAGCGGAGsequence 90 shRNA ATGGCAGGAAGAAGCGGAGTTCAAGAGACTCCGCTTCTTC sequenceCTGCCATTTTTT 91 H1 promoter GAACGCTGACGTCATCAACCCGCTCCAAGGAATCGCGGGCand shRT CCAGTGTCACTAGGCGGGAACACCCAGCGCGCGTGCGCCC sequenceTGGCAGGAAGATGGCTGTGAGGGACAGGGGAGTGGCGCCCTGCAATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGACCACTTGGATCCGCGGAGACAGCGACGAAGAGCTTCAAGAGAGCTCTTCGTCGCTGTCTCCGCTTTTT 92 H1 CCR5GAACGCTGACGTCATCAACCCGCTCCAAGGAATCGCGGGC sequenceCCAGTGTCACTAGGCGGGAACACCCAGCGCGCGTGCGCCCTGGCAGGAAGATGGCTGTGAGGGACAGGGGAGTGGCGCCCTGCAATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGACCACTTGGATCCGTGTCAAGTCCAATCTATGTTCAAGAGACATAGATTGGACTTGACACTTTTT 93 Primer AGGAATTGATGGCGAGAAGG 94Primer CCCCAAAGAAGGTCAAGGTAATCA 95 Primer AGCGCGGCTACAGCTTCA 96 PrimerGGCGACGTAGCACAGCTTCT 97 AGT103 TGTAAACTGAGCTTGCTCTA CCR5 miR30 98AGT103-R5- TGTAAACTGAGCTTGCTCGC 1 99 AGT103-R5- CATAGATTGGACTTGACAC 2100 CAG TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATA promoterGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG 101 H1 elementGAACGCTGACGTCATCAACCCGCTCCAAGGAATCGCGGGCCCAGTGTCACTAGGCGGGAACACCCAGCGCGCGTGCGCCCTGGCAGGAAGATGGCTGTGAGGGACAGGGGAGTGGCGCCCTGCAATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTC TGTATGAGACCACTT 102 3′ LTRTGGAAGGGCTAATTCACTCCCAACGAAGATAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCAT GTCA 103 7SK promoterCTGCAGTATTTAGCATGCCCCACCCATCTGCAAGGCATTCTGGATAGTGTCAAAACAGCCGGAAATCAAGTCCGTTTATCTCAAACTTTAGCATTTTGGGAATAAATGATATTTGCTATGCTGGTTAAATTAGATTTTAGTTAAATTTCCTGCTGAAGCTCTAGTACGATAAGCAACTTGACCTAAGTGTAAAGTTGAGATTTCCTTCAGGTTTATATAGCTTGTGCGCCGCCTGGCTACCTC 104 miR155 TatCTGGAGGCTTGCTGAAGGCTGTATGCTGTCCGCTTCTTCCTGCCATAGGGTTTTGGCCACTGACTGACCCTATGGGGAAGAAGCGGACAGGACACAAGGCCTGTTACTAGCACTCACATGG AACAAATGGCC 105 pRSV RevAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGAATTCGATGTACGGGCCAGATATACGCGTATCTGAGGGGACTAGGGTGTGTTTAGGCGAAAAGCGGGGCTTCGGTTGTACGCGGTTAGGAGTCCCCTCAGGATATAGTAGTTTCGCTTTTGCATAGGGAGGGGGAAATGTAGTCTTATGCAATACACTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACAGGTCTGACATGGATTGGACGAACCACTGAATTCCGCATTGCAGAGATAATTGTATTTAAGTGCCTAGCTCGATACAATAAACGCCATTTGACCATTCACCACATTGGTGTGCACCTCCAAGCTCGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCCCTCGAAGCTAGTCGATTAGGCATCTCCTATGGCAGGAAGAAGCGGAGACAGCGACGAAGACCTCCTCAAGGCAGTCAGACTCATCAAGTTTCTCTATCAAAGCAACCCACCTCCCAATCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCCTTAGCACTTATCTGGGACGATCTGCGGAGCCTGTGCCTCTTCAGCTACCACCGCTTGAGAGACTTACTCTTGATTGTAACGAGGATTGTGGAACTTCTGGGACGCAGGGGGTGGGAAGCCCTCAAATATTGGTGGAATCTCCTACAATATTGGAGTCAGGAGCTAAAGAATAGTGCTGTTAGCTTGCTCAATGCCACAGCTATAGCAGTAGCTGAGGGGACAGATAGGGTTATAGAAGTAGTACAAGAAGCTTGGCACTGGCCGTCGTTTTACATGATCTGAGCCTGGGAGATCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCACAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGC GGAAG 106 pCMV-VSV-GAGCTTGGCCCATTGCATACGTTGTATCCATATCATAATAT GGTACATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGGTCGACCGATCCTGAGAACTTCAGGGTGAGTTTGGGGACCCTTGATTGTTCTTTCTTTTTCGCTATTGTAAAATTCATGTTATATGGAGGGGGCAAAGTTTTCAGGGTGTTGTTTAGAATGGGAAGATGTCCCTTGTATCACCATGGACCCTCATGATAATTTTGTTTCTTTCACTTTCTACTCTGTTGACAACCATTGTCTCCTCTTATTTTCTTTTCATTTTCTGTAACTTTTTCGTTAAACTTTAGCTTGCATTTGTAACGAATTTTTAAATTCACTTTTGTTTATTTGTCAGATTGTAAGTACTTTCTCTAATCACTTTTTTTTCAAGGCAATCAGGGTATATTATATTGTACTTCAGCACAGTTTTAGAGAACAATTGTTATAATTAAATGATAAGGTAGAATATTTCTGCATATAAATTCTGGCTGGCGTGGAAATATTCTTATTGGTAGAAACAACTACACCCTGGTCATCATCCTGCCTTTCTCTTTATGGTTACAATGATATACACTGTTTGAGATGAGGATAAAATACTCTGAGTCCAAACCGGGCCCCTCTGCTAACCATGTTCATGCCTTCTTCTCTTTCCTACAGCTCCTGGGCAACGTGCTGGTTGTTGTGCTGTCTCATCATTTTGGCAAAGAATTCCTCGACGGATCCGCCATGAAGTGCCTTTTGTACTTAGCCTTTTTATTCATTGGGGTGAATTGCAAGTTCACCATAGTTTTTCCACACAACCAAAAAGGAAACTGGAAAAATGTTCCTTCTAATTACCATTATTGCCCGTCAAGCTCAGATTTAAATTGGCATAATGACTTAATAGGCACAGCCTTACAAGTCAAAATGCCCAAGAGTCACAAGGCTATTCAAGCAGACGGTTGGATGTGTCATGCTTCCAAATGGGTCACTACTTGTGATTTCCGCTGGTATGGACCGAAGTATATAACACATTCCATCCGATCCTTCACTCCATCTGTAGAACAATGCAAGGAAAGCATTGAACAAACGAAACAAGGAACTTGGCTGAATCCAGGCTTCCCTCCTCAAAGTTGTGGATATGCAACTGTGACGGATGCCGAAGCAGTGATTGTCCAGGTGACTCCTCACCATGTGCTGGTTGATGAATACACAGGAGAATGGGTTGATTCACAGTTCATCAACGGAAAATGCAGCAATTACATATGCCCCACTGTCCATAACTCTACAACCTGGCATTCTGACTATAAGGTCAAAGGGCTATGTGATTCTAACCTCATTTCCATGGACATCACCTTCTTCTCAGAGGACGGAGAGCTATCATCCCTGGGAAAGGAGGGCACAGGGTTCAGAAGTAACTACTTTGCTTATGAAACTGGAGGCAAGGCCTGCAAAATGCAATACTGCAAGCATTGGGGAGTCAGACTCCCATCAGGTGTCTGGTTCGAGATGGCTGATAAGGATCTCTTTGCTGCAGCCAGATTCCCTGAATGCCCAGAAGGGTCAAGTATCTCTGCTCCATCTCAGACCTCAGTGGATGTAAGTCTAATTCAGGACGTTGAGAGGATCTTGGATTATTCCCTCTGCCAAGAAACCTGGAGCAAAATCAGAGCGGGTCTTCCAATCTCTCCAGTGGATCTCAGCTATCTTGCTCCTAAAAACCCAGGAACCGGTCCTGCTTTCACCATAATCAATGGTACCCTAAAATACTTTGAGACCAGATACATCAGAGTCGATATTGCTGCTCCAATCCTCTCAAGAATGGTCGGAATGATCAGTGGAACTACCACAGAAAGGGAACTGTGGGATGACTGGGCACCATATGAAGACGTGGAAATTGGACCCAATGGAGTTCTGAGGACCAGTTCAGGATATAAGTTTCCTTTATACATGATTGGACATGGTATGTTGGACTCCGATCTTCATCTTAGCTCAAAGGCTCAGGTGTTCGAACATCCTCACATTCAAGACGCTGCTTCGCAACTTCCTGATGATGAGAGTTTATTTTTTGGTGATACTGGGCTATCCAAAAATCCAATCGAGCTTGTAGAAGGTTGGTTCAGTAGTTGGAAAAGCTCTATTGCCTCTTTTTTCTTTATCATAGGGTTAATCATTGGACTATTCTTGGTTCTCCGAGTTGGTATCCATCTTTGCATTAAATTAAAGCACACCAAGAAAAGACAGATTTATACAGACATAGAGATGAACCGACTTGGAAAGTGATAAGGATCCGTCGAGGAATTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATCCCTCGACGGATCGGCCGCAATTCGTAATCATGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGAGCTCCACCGCGGTGGCGGCCGCTCTAGA 107 PSPAX2 deltaGTCGACATTGATTATTGACTAGTTATTAATAGTAATCAATT RevACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTCGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTAAAGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGGCGGTCGGGCTGTAACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTGCGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCATCTCCAGCCTCGGGGCTGCCGCAGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTCGGGCCGGCCGCGTTGACGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGAAAAAAGCACAGCAAGCAGCAGCTGACACAGGACACAGCAATCAGGTCAGCCAAAATTACCCTATAGTGCAGAACATCCAGGGGCAAATGGTACATCAGGCCATATCACCTAGAACTTTAAATGCATGGGTAAAAGTAGTAGAAGAGAAGGCTTTCAGCCCAGAAGTGATACCCATGTTTTCAGCATTATCAGAAGGAGCCACCCCACAAGATTTAAACACCATGCTAAACACAGTGGGGGGACATCAAGCAGCCATGCAAATGTTAAAAGAGACCATCAATGAGGAAGCTGCAGAATGGGATAGAGTGCATCCAGTGCATGCAGGGCCTATTGCACCAGGCCAGATGAGAGAACCAAGGGGAAGTGACATAGCAGGAACTACTAGTACCCTTCAGGAACAAATAGGATGGATGACACATAATCCACCTATCCCAGTAGGAGAAATCTATAAAAGATGGATAATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTACCAGCATTCTGGACATAAGACAAGGACCAAAGGAACCCTTTAGAGACTATGTAGACCGATTCTATAAAACTCTAAGAGCCGAGCAAGCTTCACAAGAGGTAAAAAATTGGATGACAGAAACCTTGTTGGTCCAAAATGCGAACCCAGATTGTAAGACTATTTTAAAAGCATTGGGACCAGGAGCGACACTAGAAGAAATGATGACAGCATGTCAGGGAGTGGGGGGACCCGGCCATAAAGCAAGAGTTTTGGCTGAAGCAATGAGCCAAGTAACAAATCCAGCTACCATAATGATACAGAAAGGCAATTTTAGGAACCAAAGAAAGACTGTTAAGTGTTTCAATTGTGGCAAAGAAGGGCACATAGCCAAAAATTGCAGGGCCCCTAGGAAAAAGGGCTGTTGGAAATGTGGAAAGGAAGGACACCAAATGAAAGATTGTACTGAGAGACAGGCTAATTTTTTAGGGAAGATCTGGCCTTCCCACAAGGGAAGGCCAGGGAATTTTCTTCAGAGCAGACCAGAGCCAACAGCCCCACCAGAAGAGAGCTTCAGGTTTGGGGAAGAGACAACAACTCCCTCTCAGAAGCAGGAGCCGATAGACAAGGAACTGTATCCTTTAGCTTCCCTCAGATCACTCTTTGGCAGCGACCCCTCGTCACAATAAAGATAGGGGGGCAATTAAAGGAAGCTCTATTAGATACAGGAGCAGATGATACAGTATTAGAAGAAATGAATTTGCCAGGAAGATGGAAACCAAAAATGATAGGGGGAATTGGAGGTTTTATCAAAGTAGGACAGTATGATCAGATACTCATAGAAATCTGCGGACATAAAGCTATAGGTACAGTATTAGTAGGACCTACACCTGTCAACATAATTGGAAGAAATCTGTTGACTCAGATTGGCTGCACTTTAAATTTTCCCATTAGTCCTATTGAGACTGTACCAGTAAAATTAAAGCCAGGAATGGATGGCCCAAAAGTTAAACAATGGCCATTGACAGAAGAAAAAATAAAAGCATTAGTAGAAATTTGTACAGAAATGGAAAAGGAAGGAAAAATTTCAAAAATTGGGCCTGAAAATCCATACAATACTCCAGTATTTGCCATAAAGAAAAAAGACAGTACTAAATGGAGAAAATTAGTAGATTTCAGAGAACTTAATAAGAGAACTCAAGATTTCTGGGAAGTTCAATTAGGAATACCACATCCTGCAGGGTTAAAACAGAAAAAATCAGTAACAGTACTGGATGTGGGCGATGCATATTTTTCAGTTCCCTTAGATAAAGACTTCAGGAAGTATACTGCATTTACCATACCTAGTATAAACAATGAGACACCAGGGATTAGATATCAGTACAATGTGCTTCCACAGGGATGGAAAGGATCACCAGCAATATTCCAGTGTAGCATGACAAAAATCTTAGAGCCTTTTAGAAAACAAAATCCAGACATAGTCATCTATCAATACATGGATGATTTGTATGTAGGATCTGACTTAGAAATAGGGCAGCATAGAACAAAAATAGAGGAACTGAGACAACATCTGTTGAGGTGGGGATTTACCACACCAGACAAAAAACATCAGAAAGAACCTCCATTCCTTTGGATGGGTTATGAACTCCATCCTGATAAATGGACAGTACAGCCTATAGTGCTGCCAGAAAAGGACAGCTGGACTGTCAATGACATACAGAAATTAGTGGGAAAATTGAATTGGGCAAGTCAGATTTATGCAGGGATTAAAGTAAGGCAATTATGTAAACTTCTTAGGGGAACCAAAGCACTAACAGAAGTAGTACCACTAACAGAAGAAGCAGAGCTAGAACTGGCAGAAAACAGGGAGATTCTAAAAGAACCGGTACATGGAGTGTATTATGACCCATCAAAAGACTTAATAGCAGAAATACAGAAGCAGGGGCAAGGCCAATGGACATATCAAATTTATCAAGAGCCATTTAAAAATCTGAAAACAGGAAAATATGCAAGAATGAAGGGTGCCCACACTAATGATGTGAAACAATTAACAGAGGCAGTACAAAAAATAGCCACAGAAAGCATAGTAATATGGGGAAAGACTCCTAAATTTAAATTACCCATACAAAAGGAAACATGGGAAGCATGGTGGACAGAGTATTGGCAAGCCACCTGGATTCCTGAGTGGGAGTTTGTCAATACCCCTCCCTTAGTGAAGTTATGGTACCAGTTAGAGAAAGAACCCATAATAGGAGCAGAAACTTTCTATGTAGATGGGGCAGCCAATAGGGAAACTAAATTAGGAAAAGCAGGATATGTAACTGACAGAGGAAGACAAAAAGTTGTCCCCCTAACGGACACAACAAATCAGAAGACTGAGTTACAAGCAATTCATCTAGCTTTGCAGGATTCGGGATTAGAAGTAAACATAGTGACAGACTCACAATATGCATTGGGAATCATTCAAGCACAACCAGATAAGAGTGAATCAGAGTTAGTCAGTCAAATAATAGAGCAGTTAATAAAAAAGGAAAAAGTCTACCTGGCATGGGTACCAGCACACAAAGGAATTGGAGGAAATGAACAAGTAGATGGGTTGGTCAGTGCTGGAATCAGGAAAGTACTATTTTTAGATGGAATAGATAAGGCCCAAGAAGAACATGAGAAATATCACAGTAATTGGAGAGCAATGGCTAGTGATTTTAACCTACCACCTGTAGTAGCAAAAGAAATAGTAGCCAGCTGTGATAAATGTCAGCTAAAAGGGGAAGCCATGCATGGACAAGTAGACTGTAGCCCAGGAATATGGCAGCTAGATTGTACACATTTAGAAGGAAAAGTTATCTTGGTAGCAGTTCATGTAGCCAGTGGATATATAGAAGCAGAAGTAATTCCAGCAGAGACAGGGCAAGAAACAGCATACTTCCTCTTAAAATTAGCAGGAAGATGGCCAGTAAAAACAGTACATACAGACAATGGCAGCAATTTCACCAGTACTACAGTTAAGGCCGCCTGTTGGTGGGCGGGGATCAAGCAGGAATTTGGCATTCCCTACAATCCCCAAAGTCAAGGAGTAATAGAATCTATGAATAAAGAATTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTTAAGACAGCAGTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTTGGAAAGGACCAGCAAAGCTCCTCTGGAAAGGTGAAGGGGCAGTAGTAATACAAGATAATAGTGACATAAAAGTAGTGCCAAGAAGAAAAGCAAAGATCATCAGGGATTATGGAAAACAGATGGCAGGTGATGATTGTGTGGCAAGTAGACAGGATGAGGATTAACACATGGAATTCTGCAACAACTGCTGTTTATCCATTTCAGAATTGGAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTGCTAGCAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCATATGCTGGCTGCCATGAACAAAGGTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGAAGATCCCTCGACCTGCAGCCCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCGGATCCGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCCGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCG CGCACATTTCCCCGAAAAGTGCCACCTG

While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention.

What is claimed is:
 1. A method of treating cells infected with HIV, themethod comprising: (a) contacting peripheral blood mononuclear cells(PBMC) isolated from a subject infected with HIV with a therapeuticallyeffective amount of a stimulatory agent, wherein the contacting iscarried out ex vivo; (b) transducing the PBMC ex vivo with a viraldelivery system encoding at least one genetic element, wherein the atleast one genetic element comprises a small RNA capable of inhibitingproduction of chemokine receptor CCR5 or at least one small RNA capableof targeting an HIV RNA sequence; and (c) culturing the transduced PBMCfor at least 1 day.
 2. The method of claim 1, further comprisingpositively selecting HIV-specific CD4+ T cells from the PBMC.
 3. Themethod of claim 2, wherein the HIV-specific CD4+ T cells are positivelyselected using at least one physical method of selection.
 4. The methodof claim 1, further comprising infusing the transduced PBMC into asubject.
 5. The method of claim 4, wherein the subject is a human. 6.The method of claim 1, wherein the stimulatory agent comprises apeptide.
 7. The method of claim 6, wherein the peptide comprises a gagpeptide.
 8. The method of claim 1, wherein the stimulatory agentcomprises a vaccine.
 9. The method of claim 8, wherein the vaccinecomprises a HIV vaccine.
 10. The method of claim 9, wherein the HIVvaccine comprises a MVA/HIV62B vaccine or a variant thereof.
 11. Themethod of claim 1, wherein the at least one genetic element comprises asmall RNA capable of inhibiting production of chemokine receptor CCR5and at least one small RNA capable of targeting an HIV RNA sequence. 12.The method of claim 1 or 11, wherein the HIV RNA sequence comprises aHIV Vif sequence, a HIV Tat sequence, or a variant thereof.
 13. Themethod of claim 1 or 11, wherein the at least one genetic elementcomprises a microRNA or a shRNA.
 14. The method of claim 13, wherein theat least one genetic element comprises a microRNA cluster.
 15. Themethod of claim 13, wherein the at least one genetic element comprises amicroRNA having at least 80%, or at least 85%, or at least 90%, or atleast 95% percent identity with SEQ ID NO:
 1. 16. The method of claim13, wherein the at least one genetic element comprises SEQ ID NO:
 1. 17.The method of claim 13, wherein the at least one genetic elementcomprises a microRNA having: a. at least 80%, or at least 85%, or atleast 90%, or at least 95% percent identity with SEQ ID NO: 2; or b. atleast 80%, or at least 85%, or at least 90%, or at least 95% percentidentity with SEQ ID NO:
 3. 18. The method of claim 13, wherein the atleast one genetic element comprises SEQ ID NO: 2; or SEQ ID NO:
 3. 19.The method of claim 14, wherein the microRNA cluster comprises asequence having at least 80%, or at least 85%, or at least 90%, or atleast 95% percent identity with SEQ ID NO:
 31. 20. The method of claim14, wherein the microRNA cluster comprises SEQ ID NO:
 31. 21. A methodof treating HIV infection in a subject, the method comprising: (a)immunizing the subject with an effective amount of a first stimulatoryagent; (b) removing leukocytes from the subject and purifying peripheralblood mononuclear cells (PBMC); (c) contacting the PBMC ex vivo with atherapeutically effective amount of a second stimulatory agent; (d)transducing the PBMC ex vivo with a viral delivery system encoding atleast one genetic element; and (e) culturing the transduced PBMC for atleast 1 day.
 22. The method of claim 21, further comprising positivelyselecting HIV-specific CD4+ T cells from the PBMC.
 23. The method ofclaim 22, wherein the HIV-specific CD4+ T cells are positively selectedusing at least one physical method of selection.
 24. The method of claim21, further comprising infusing the transduced PBMC into the subject.25. The method of claim 21, wherein the first and second stimulatoryagents are the same.
 26. The method of claim 21, wherein at least one ofthe first and second stimulatory agents comprises a HIV vaccine.
 27. Themethod of claim 26, wherein the HIV vaccine comprises a MVA/HIV62Bvaccine or a variant thereof.
 28. The method of claim 21, wherein theviral delivery system comprises a lentiviral particle.
 29. The method ofclaim 21, wherein the at least one genetic element comprises a small RNAcapable of inhibiting production of chemokine receptor CCR5 or at leastone small RNA capable of targeting an HIV RNA sequence.
 30. A method oftreating HIV infection in a subject, the method comprising: (a)immunizing the subject with an effective amount of a first stimulatoryagent; (b) removing leukocytes from the subject and purifying peripheralblood mononuclear cells (PBMC); (c) positively selecting HIV-specificCD4+ T cells from the PBMC; (d) contacting the HIV-specific CD4+ T cellsex vivo with a therapeutically effective amount of a second stimulatoryagent; (d) transducing the HIV-specific CD4+ T cells ex vivo with aviral delivery system encoding at least one genetic element; and (e)culturing the transduced PBMC for at least 1 day.